Death domain containing receptors

ABSTRACT

The present invention relates to novel Death Domain Containing Receptor (DR3 and DR3-V1) proteins that are members of the tumor necrosis factor (TNF) receptor family. In particular, isolated nucleic acid molecules are provided encoding the human DR3 and DR3-V1 proteins. DR3 and DR3-V1 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of DR3 and DR3-V1 activity.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application, which claims benefit under 35 U.S.C. § 119(e)of U.S. Provisional Application Nos. 60/314,314 and 60/303,155 filed onAug. 24, 2001 and Jul. 6, 2001 respectively, is a Continuation-In-Partof, and claim benefit under 35 U.S.C. § 120 to, U.S. patent applicationSer. No. 09/557,908 filed on Apr. 21, 2000; which claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Application Nos. 60/136,741 and60/130,488 filed on May 28, 1999 and Apr. 22, 1999 respectively; whichin turn is a Continuation-In-Part of, and claims benefit under 35 U.S.C.§ 120 of U.S. patent application Ser. No. 08/815,469 filed on Mar. 11,1997 (now U.S. Pat. No. 6,153,402, issued Nov. 28, 2000); which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos.60/037,341, 60/028,711 and 60/013,285 filed on Feb. 6, 1997, Oct. 17,1996 and Mar. 12, 1996 respectively.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a novel member of the tumornecrosis factor family of receptors. More specifically, isolated nucleicacid molecules are provided encoding human Death Domain ContainingReceptors (DR3 and DR3-V1). Death Domain Containing Receptorpolypeptides are also provided, as are vectors, host cells andrecombinant methods for producing the same. The invention furtherrelates to screening methods for identifying agonists and antagonists ofDR3 activity.

[0004] 2. Related Art

[0005] Many biological actions, for instance, response to certainstimuli and natural biological processes, are controlled by factors,such as cytokines. Many cytokines act through receptors by engaging thereceptor and producing an intra-cellular response.

[0006] For example, tumor necrosis factors (TNF) alpha and beta arecytokines which act through TNF receptors to regulate numerousbiological processes, including protection against infection andinduction of shock and inflammatory disease. The TNF molecules belong tothe “TNF-ligand” superfamily, and act together with their receptors orcounter-ligands, the “TNF-receptor” superfamily. So far, nine members ofthe TNF ligand superfamily have been identified and ten members of theTNF-receptor superfamily have been characterized.

[0007] Among the ligands there are included TNF-α, lymphotoxin-α (LT-α,also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β),FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).The superfamily of TNF receptors includes the p55TNF receptor, p75TNFreceptor, TNF receptor-related protein, FAS antigen or APO-1, CD40,CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (A. Meager,Biologicals, 22:291-295 (1994)).

[0008] Many members of the TNF-ligand superfamily are expressed byactivated T-cells, implying that they are necessary for T-cellinteractions with other cell types, which underlie cell ontogeny andfunctions. (A. Meager, supra).

[0009] Considerable insight into the essential functions of severalmembers of the TNF receptor family has been gained from theidentification and creation of mutants that abolish the expression ofthese proteins. For example, naturally occurring mutations in the FASantigen and its ligand cause lymphoproliferative disease (R.Watanabe-Fukunaga et al., Nature 356:314 (1992)), perhaps reflecting afailure of programmed cell death. Mutations of the CD40 ligand cause anX-linked immunodeficiency state characterized by high levels ofimmunoglobulin M and low levels of immunoglobulin G in plasma,indicating faulty T-cell-dependent B-cell activation (R. C. Allen etal., Science 259:990 (1993)). Targeted mutations of the low affinitynerve growth factor receptor cause a disorder characterized by faultysensory innovation of peripheral structures (K. F. Lee et al., Cell69:737 (1992)).

[0010] TNF and LT-α are capable of binding to two TNF receptors (the 55-and 75-kd TNF receptors). A large number of biological effects elicitedby TNF and LT-α, acting through their receptors, include hemorrhagicnecrosis of transplanted tumors, cytotoxicity, a role in endotoxicshock, inflammation, immunoregulation, proliferation and anti-viralresponses, as well as protection against the deleterious effects ofionizing radiation. TNF and LT-α are involved in the pathogenesis of awide range of diseases, including endotoxic shock, cerebral malaria,tumors, autoimmune disease, AIDS and graft-host rejection (B. Beutlerand C. Von Huffel, Science 264:667-668 (1994)). Mutations in the p55receptor cause increased susceptibility to microbial infection.

[0011] Moreover, an about 80 amino acid domain near the C-terminus ofTNFR1 (p55) and Fas was reported as the “death domain,” which isresponsible for transducing signals for programmed cell death (Tartagliaet al., Cell 74:845 (1993)).

[0012] Apoptosis, or programmed cell death, is a physiologic processessential to the normal development and homeostasis of multicellularorganisms (H. Steller, Science 267, 1445-1449 (1995)). Derangements ofapoptosis contribute to the pathogenesis of several human diseasesincluding cancer, neurodegenerative disorders, and acquired immunedeficiency syndrome (C. B. Thompson, Science 267, 1456-1462 (1995)).Recently, much attention has focused on the signal transduction andbiological function of two cell surface death receptors, Fas/APO-1 andTNFR-1 (J. L. Cleveland et al., Cell 81,479-482 (1995); A. Fraser etal., Cell 85, 781-784 (1996); S. Nagata et al., Science 267, 1449-56(1995)). Both are members of the TNF receptor family which also includeTNFR-2, low affinity NGFR, CD40, and CD30, among others (C. A. Smith etal., Science 248, 1019-23 (1990); M. Tewari et al., in Modular Texts inMolecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman andHall, London, 1995). While family members are defined by the presence ofcysteine-rich repeats in their extracellular domains, Fas/APO-1 andTNFR-1 also share a region of intracellular homology, appropriatelydesignated the “death domain,” which is distantly related to theDrosophila suicide gene, reaper (P. Golstein et al., Cell 81, 185-6(1995); K. White et al., Science 264,677-83 (1994)). This shared deathdomain suggests that both receptors interact with a related set ofsignal transducing molecules that, until recently, remainedunidentified. Activation of Fas/APO-1 recruits the deathdomain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan et al.,Cell 81: 505-12 (1995); M. P. Boldin et al., J. Biol Chem 270: 7795-8(1995); F. C. Kischkel et al., EMBO 14: 5579-5588 (1995)), which in turnbinds and presumably activates FLICE/MACH1, a member of the ICE/CED-3family of pro-apoptotic proteases (M. Muzio et al., Cell 85: 817-827(1996); M. P. Boldin et al., Cell 85: 803-815 (1996)). While the centralrole of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an arrayof diverse biological activities—many of which stem from its ability toactivate NF-kB (L. A. Tartaglia et al., Immunol Today 13: 151-3 (1992)).Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD,which like FADD, also contains a death domain (H. Hsu et al., Cell 81:495-504 (1995); H. Hsu et al., Cell 84: 299-308 (1996)). Through itsassociations with a number of signaling molecules including FADD, TRAF2,and RIP, TRADD can signal both apoptosis and NF-kB activation, Id.; H.Hsu et al., Immunity 4: 387-396 (1996)).

[0013] The effects of TNF family ligands and TNF family receptors arevaried and influence numerous functions, both normal and abnormal, inthe biological processes of the mammalian system. There is a clear need,therefore, for identification and characterization of such receptors andligands that influence biological activity, both normally and in diseasestates. In particular, there is a need to isolate and characterize novelmembers of the TNF receptor family.

SUMMARY OF THE INVENTION

[0014] The present invention provides for isolated nucleic acidmolecules comprising, or alternatively consisting of, nucleic acidsequences encoding the amino acid sequences shown in SEQ ID NO:2 and SEQID NO:4 or the amino acid sequence encoding the cDNAs deposited as ATCCDeposit No. 97456 on Mar. 1, 1996 and ATCC Deposit No. 97757 on Oct. 10,1996.

[0015] The present invention also provides vectors and host cells forrecombinant expression of the nucleic acid molecules described herein,as well as to methods of making such vectors and host cells and forusing them for production of DR3 or DR3 Variant 1 (DR3-V 1) (formerlynamed DDCR) polypeptides or peptides by recombinant techniques.

[0016] The invention further provides an isolated DR3 or DR3-V1polypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

[0017] The present invention also provides diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR3 or DR3-V1protein. Thus, for instance, a diagnostic assay in accordance with theinvention for detecting over-expression of DR3 or DR3-V1, or solubleform thereof, compared to normal control tissue samples may be used todetect the presence of tumors.

[0018] Tumor Necrosis Factor (TNF) family ligands are known to be amongthe most pleiotropic cytokines, inducing a large number of cellularresponses, including cytotoxicity, anti-viral activity, immunoregulatoryactivities, and the transcriptional regulation of several genes.Cellular response to TNF-family ligands include not only normalphysiological responses, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Apoptosis-programmed celldeath—is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes. Diseases associatedwith increased cell survival, or the inhibition of apoptosis, includecancers, autoimmune disorders, viral infections, inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS,neurodegenerative disorders, myelodysplastic syndromes, ischemic injury,toxin-induced liver disease, septic shock, cachexia and anorexia.

[0019] Thus, the invention further provides a method for enhancingapoptosis induced by a TNF-family ligand, which involves administeringto a cell which expresses the DR3 polypeptide an effective amount of anagonist capable of increasing DR3 mediated signaling. Preferably, DR3mediated signaling is increased to treat and/or prevent a diseasewherein decreased apoptosis is exhibited. Examples of such diseasesinclude, but are not limited to, graft vs. host disease (acute and/orchronic), multiple sclerosis, Sjogren's syndrome, Grave's disease,Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,Behcet's disease, Crohn's disease, polymyositis, systemic lupuserythematosus, immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, rheumatoid arthritis and ulcerative colitis.

[0020] In a further aspect, the present invention is directed to amethod for inhibiting apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell which expresses the DR3 polypeptide aneffective amount of an antagonist capable of decreasing DR3 mediatedsignaling. Preferably, DR3 mediated signaling is decreased to treatand/or prevent a disease wherein increased apoptosis is exhibited.

[0021] Whether any candidate “agonist” or “antagonist” of the presentinvention can enhance or inhibit apoptosis can be determined usingart-known TNF-family ligand/receptor cellular response assays, includingthose described in more detail below. Thus, in a further aspect, ascreening method is provided for determining whether a candidate agonistor antagonist is capable of enhancing or inhibiting a cellular responseto a TNF-family ligand. The method involves contacting cells whichexpress the DR3 or DR3-V1 polypeptide with a candidate compound and aTNF-family ligand, assaying a cellular response, and comparing thecellular response to a standard cellular response, the standard beingassayed when contact is made with the ligand in absence of the candidatecompound, whereby an increased cellular response over the standardindicates that the candidate compound is an agonist of theligand/receptor signaling pathway and a decreased cellular responsecompared to the standard indicates that the candidate compound is anantagonist of the ligand/receptor signaling pathway. By the invention, acell expressing the DR3 or DR3-V1 polypeptide can be contacted witheither an endogenous or exogenously administered TNF-family ligand.

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIGS. 1A-1C (SEQ ID NOs:1 and 2) shows the nucleotide and deducedamino acid sequence of DR3-V1. It is predicted that amino acids 1-35constitute the signal peptide, amino acids 36-212 constitute theextracellular domain, amino acids 213-235 constitute the transmembranedomain, amino acids 236-428 constitute the intracellular domain, andamino acids 353-419 the death domain.

[0023] FIGS. 2A-2B (SEQ ID NOs:3 and 4) shows the nucleotide and deducedamino acid sequence of DR3. It is predicted that amino acids 1-24constitute the signal peptide, amino acids 25-201 constitute theextracellular domain, amino acids 202-224 constitute the transmembranedomain, amino acids 225-417 constitute the intracellular domain, andamino acids 342-408 constitute the death domain.

[0024] FIGS. 3A-3D shows the regions of similarity between the aminoacid sequences of the DR3-V1, human tumor necrosis factor receptor 1,and Fas receptor (SEQ ID NOs:5 and 6).

[0025]FIG. 4 shows an analysis of the DR3-V1 amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 1-22, 33-56, 59-82, 95-112, 122-133, 161-177,179-190, 196-205 in SEQ ID NO:2 correspond to the shown highly antigenicregions of the DR3-V1 protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides isolated nucleic acid moleculescomprising, or alternatively consisting of, a nucleic acid sequenceencoding the DR3-V1 or DR3 polypeptide whose amino acid sequence isshown in SEQ ID NO:2 and SEQ ID NO:4, respectively, or a fragment of thepolypeptide. The DR3-V1 and DR3 polypeptides of the present inventionshare sequence homology with human TNF RI and Fas (FIG. 4). Thenucleotide sequence shown in SEQ ID NO:1 was obtained by sequencing theHTTNB61 clone, which was deposited on Mar. 1, 1996 at the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA, and given Accession Number 97456. The deposited cDNA is containedin the pBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.). Thenucleotide sequence shown in SEQ ID NO:3 was obtained by sequencing acDNA obtained from a HUVEC library, which was deposited on Oct. 10, 1996at the American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209, USA, and given Accession Number 97757. Thedeposited cDNA is contained in the pBluescript SK(−) plasmid(Stratagene, LaJolla, Calif.).

[0027] Nucleic Acid Molecules

[0028] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0029] By “isolated” polypeptide or protein is intended a polypeptide orprotein removed from its native environment. For example, recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for purposes of the invention, as are native orrecombinant polypeptides which have been substantially purified by anysuitable technique such as, for example, the single-step purificationmethod disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0030] Using the information provided herein, such as the nucleic acidsequence set out in SEQ ID NO:1 or SEQ ID NO:3, a nucleic acid moleculeof the present invention encoding a DR3-V1 or DR3 polypeptide may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in SEQ ID NO:1 wasdiscovered in a cDNA library derived from cells of a human testis tumor.Also illustrative of the invention, the nucleic acid molecule describedin SEQ ID NO:3 was discovered in a human HUVEC cDNA library. Inaddition, the genes of the present invention have also been identifiedin cDNA libraries of the following tissues: fetal liver, fetal brain,tonsil and leukocyte. Furthermore, multiple forms of DR3 transcript areseen in Northern Blots and PCR reactions indicating that multiplevariants of the transcript exists, possibly due to alternate splicing ofthe message.

[0031] The DR3-V1 (formerly called DDCR) gene contains an open readingframe encoding a protein of about 428 amino acid residues whoseinitiation codon is at position 198-200 of the nucleotide sequence shownin SEQ ID NO.1, with a leader sequence of about 35 amino acid residues,and a deduced molecular weight of about 47 kDa. Of known members of theTNF receptor family, the DR3-V1 polypeptide of the invention shares thegreatest degree of homology with human TNF R1. The DR3-V1 polypeptideshown in SEQ ID NO:2 is about 20% identical and about 50% similar tohuman TNF R1.

[0032] The DR3 gene contains an open reading frame encoding a protein ofabout 417 amino acid residues whose initiation codon is at position 1-3of the nucleotide sequence shown in SEQ ID NO:3, with a leader sequenceof about 24 amino acid residues, and a deduced molecular weight of about43 kDa. Of known members of the TNF receptor family, the DR3 polypeptideof the invention shares the greatest degree of homology with human TNFR1. The DR3 polypeptide shown in SEQ ID NO:3 is about 20% identical andabout 50% similar to human TNF R1.

[0033] As indicated, the present invention also provides the matureform(s) of the DR3-V1 and DR3 protein of the present invention.According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretory leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that the cleavage specificity of asecreted protein is ultimately determined by the primary structure ofthe complete protein, that is, it is inherent in the amino acid sequenceof the polypeptide. Therefore, the present invention provides anucleotide sequence encoding the mature DR3-V1 or DR3 polypeptideshaving the amino acid sequence encoded by the cDNAs contained in thehost identified as ATCC Deposit No. 97456 or 97757, respectively, and asshown in SEQ ID NO:2 and SEQ ID NO:4. By the mature DR3-V1 or DR3protein having the amino acid sequence encoded by the cDNAs contained inthe host identified as ATCC Deposit No. 97456 or 97757, respectively, ismeant the mature form(s) of the DR3-V1 or DR3 protein produced byexpression in a mammalian cell (e.g., COS cells, as described below) ofthe complete open reading frame encoded by the human DNA sequence of thecDNA contained in the vector in the deposited host. As indicated below,the mature DR3-V1 or DR3 having the amino acid sequence encoded by thecDNAs contained in ATCC Deposit No. 97456 or 97757, respectively, may ormay not differ from the predicted “mature” DR3-V1 protein shown in SEQID NO:2 (amino acids from about 36 to about 428) or DR3 protein shown inSEQ ID NO:4 (amino acids from about 24 to about 417) depending on theaccuracy of the predicted cleavage site based on computer analysis.

[0034] Methods for predicting whether a protein has a secretory leaderas well as the cleavage point for that leader sequence are available.For instance, the method of McGeoch (Virus Res. 3:271-286 (1985)) andvon Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

[0035] In the present case, the predicted amino acid sequences of thecomplete DR3-V1 and DR3 polypeptides of the present invention wereanalyzed by a computer program (“PSORT”), see, K. Nakai and M. Kanehisa,Genomics 14:897-911 (1992)), which is an expert system for predictingthe cellular location of a protein based on the amino acid sequence. Aspart of this computational prediction of localization, the methods ofMcGeoch and von Heinje are incorporated. The analysis by the PSORTprogram predicted the cleavage sites between amino acids 35 and 36 inSEQ ID NO:2 and between amino acids 24 and 25 in SEQ ID NO:4.Thereafter, the complete amino acid sequences were further analyzed byvisual inspection, applying a simple form of the (−1 ,−3) rule of vonHeine. von Heinje, supra. Thus, the leader sequence for the DR3-V1protein is predicted to consist of amino acid residues 1- 35 in SEQ IDNO:2, while the predicted mature DR3-V1 protein consists of residues36-428. The leader sequence for the DR3 protein is predicted to consistof amino acid residues 1-24 in SEQ ID NO:4, while the predicted matureDR3 protein consists of residues 25-417.

[0036] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the actual DR3-V1 polypeptide encoded by the deposited cDNA comprisesabout 428 amino acids, but may be anywhere in the range of 410-440 aminoacids; and the actual leader sequence of this protein is about 35 aminoacids, but may be anywhere in the range of about 25 to about 45 aminoacids. The actual DR3 polypeptide encoded by the deposited cDNAcomprises about 417 amino acids, but may be anywhere in the range of400-430 amino acids; and the actual leader sequence of this protein isabout 24 amino acids, but may be anywhere in the range of about 14 toabout 34 amino acids.

[0037] As indicated, nucleic acid molecules of the present invention maybe in the form of RNA, such as mRNA, or in the form of DNA, including,for instance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA may be the coding strand, also known as the sensestrand, or it may be the non-coding strand, also referred to as theanti-sense strand.

[0038] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

[0039] However, a nucleic acid contained in a clone that is a member ofa library (e.g., a genomic or cDNA library) that has not been isolatedfrom other members of the library (e.g., in the form of a homogeneoussolution containing the clone and other members of the library) or, achromosome isolated or removed from a cell or a cell lysate (e.g., a“chromosome spread,” as in a karyotype), is not “isolated” for thepurposes of the invention. As discussed further herein, isolated nucleicacid molecules according to the present invention may be producednaturally, recombinantly, or synthetically.

[0040] Isolated nucleic acid molecules of the present invention includeDR3-V1 DNA molecules comprising, or alternatively consisting of, an openreading frame (ORF) shown in SEQ ID NO:1 and further include DNAmolecules which comprise, or alternatively consist of, a sequencesubstantially different than all or part of the ORF whose initiationcodon is at position 198-200 of the nucleotide sequence shown in SEQ IDNO:1 but which, due to the degeneracy of the genetic code, still encodethe DR3-V 1 polypeptide or a fragment thereof. Isolated nucleic acidmolecules of the present invention also include DR3 DNA moleculescomprising, or alternatively consisting of, an open reading frame (ORF)shown in SEQ ID NO:3 and further include DNA molecules which comprise,or alternatively consist of, a sequence substantially different than allor part of the ORF whose initiation codon is at position 1-3 of thenucleotide sequence shown in SEQ ID NO:3 but which, due to thedegeneracy of the genetic code, still encode the DR3 polypeptide or afragment thereof. Of course, the genetic code is well known in the art.Thus, it would be routine for one skilled in the art to generate suchdegenerate variants.

[0041] In another aspect, the invention provides isolated nucleic acidmolecules encoding the DR3-V1 polypeptide having an amino acid sequenceencoded by the cDNA contained in the plasmid deposited as ATCC DepositNo. 97456 on Mar. 1, 1996. The invention provides isolated nucleic acidmolecules encoding the DR3 polypeptide having an amino acid sequenceencoded by the cDNA contained in the plasmid deposited as ATCC DepositNo. 97757 on Oct. 10, 1996. Preferably, these nucleic acid moleculeswill encode the mature polypeptide encoded by the above-describeddeposited cDNAs. The invention further provides an isolated nucleic acidmolecule having the nucleotide sequence shown in SEQ ID NO:1 or SEQ IDNO:3 or the nucleotide sequence of the DR3-V1 or DR3 cDNA contained inthe above-described deposited plasmids, or a nucleic acid moleculehaving a sequence complementary to one of the above sequences. Suchisolated DNA molecules and fragments thereof are useful, for example, asDNA probes for gene mapping by in situ hybridization with chromosomes,and for detecting expression of the DR3-V1 or DR3 gene in human tissue(including testis tumor tissue) by Northern blot analysis.

[0042] DR3 expression has been detected in a wide range of tissues andcell types including endothelial cells, liver cells, hepatocellulartumor, lymph nodes, Hodgkin's lymphoma, tonsil, bone marrow, spleen,heart, thymus, pericardium, healing wound (skin), brain, pancreas tumor,burned skin, U937 cells, testis, colon cancer (metasticized to liver),pancreas, rejected kidney, adipose, ovary, olfactory epithelium,striatum depression, HeLa cells, LNCAP (upon treatment with +30 nMandrogen), 8 week embryo tissues, 9 week embryo tissues, fetal braintissues, fetal kidney tissues, fetal heart tissues, fetal thymustissues, fetal lung tissues, fetal liver tissues, fetal spleen tissues,T-cell helper II, activated T-cell (16 hr), activated T-cell (24 hr),primary dendritic cells, eosinophils, monocytes, keratinocytes and HUVEC(human umbilical vein endothelial cells).

[0043] The present invention is further directed to polynucleotidescomprising, or alternatively consisting of, fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of one of thedeposited cDNAs or the nucleotide sequence shown in SEQ ID NO:1 or SEQID NO:3 is intended fragments at least about 15 nt, and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. In this context“about” includes the particularly recited value and values larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides. Of course, largerfragments comprising, or alternatively consisting of, at least 50, 75,100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775,800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100,1125, 1150, 1175, 1200, 1225, 1250 or 1283 nt are also useful accordingto the present invention as are fragments corresponding to most, if notall, of the nucleotide sequence of one of the deposited cDNAs or asshown in SEQ ID NO:1 or SEQ ID NO:3. By a fragment at least 20 nt inlength, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of one of the depositedcDNAs or the nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.

[0044] The present invention is further directed to polynucleotidescomprising, or alternatively consisting of, fragments of isolatednucleic acid molecules which encode subportions of DR3-V1 and DR3. Inparticular, the invention provides polynucleotides comprising, oralternatively consisting of, the nucleotide sequences of a memberselected from the group consisting of nucleotides 198-257, 208-267,218-277, 228-287, 238-297, 248-307, 258-317, 268-327, 278-337, 288-347,298-357, 308-367, 318-377, 328-387, 338-397, 348-407, 358-417, 368-427,378-437, 388-447, 398-457, 408-469, 428-487, 458-517, 478-537, 498-557,518-577, 538-597, 558-617, 578-637, 598-657, 638-697, 658-717, 698-757,708-767, 718-767, 728-787, 738-797, 748-807, 758-817, 778-837, 788-847,808-867, 828-887, 848-907, 868-927, 888-947, 898-957, 908-967, 918-977,928-987, 948-1007, 968-1027, 988-1047, 998-1067, 1018-1077, 1038-1097,1058-1117, 1068-1127, 1088-1147, 1098-1157, 1118-1177, 1138-1197,1158-1217, 1178-1237, 1198-1257, 1218-1277, 1238-1297, 1258-1317,1278-1337, 1298-1357, 1318-1377, 1338-1397,1358-1417,1378-1437,1398-1457,1418-1477, and 1428-1481 of SEQ ID NO:1.

[0045] The present invention is further directed to polynucleotidescomprising, or alternatively consisting of, isolated nucleic acidmolecules which encode domains of DR3-V1 and DR3. In one aspect, theinvention provides polynucleotides comprising, or alternativelyconsisting of, nucleic acid molecules which encode beta-sheet regions ofDR3-V1 protein set out in Table 2. Representative examples of suchpolynucleotides include nucleic acid molecules which encode apolypeptide comprise, or alternatively consist of, one, two, three,four, five or more amino acid sequences selected from the groupconsisting of amino acid residues from about 24 to about 32, amino acidresidues from about 53 to about 58, amino acid residues from about 133to about, 142, amino acid residues from about 202 to about 234, aminoacid residues from about 281 to about 288, amino acid residues fromabout 304 to about 312, and amino acid residues from about 346 to about350 in SEQ ID NO:2. In this context “about” includes the particularlyrecited value and values larger or smaller by several (5, 4, 3, 2, or 1)amino acids. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0046] Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding one, two, three, four, five, or moreamino acids sequences selected from the group consisting of: apolypeptide comprising, or alternatively consisting of, the DR3-V1extracellular domain (amino acid residues from about 36 to about 212 inSEQ ID NO:2); a polypeptide comprising, or alternatively consisting of,the DR3-V1 transmembrane domain (amino acid residues from about 213 toabout 235 in SEQ ID NO:2; a polypeptide comprising, or alternativelyconsisting of, the DR3-V1 intracellular domain (amino acid residues fromabout 236 to about 428 in SEQ ID NO:2; and a polypeptide comprising, oralternatively consisting of, the DR3-V1 death domain (amino acidresidues from about 353 to about 419 in SEQ ID NO:2). In this context“about” includes the particularly recited value and values larger orsmaller by several (5, 4, 3, 2, or 1) amino acids. Since the location ofthese domains have been predicted by computer graphics, one of ordinaryskill would appreciate that the amino acid residues constituting thesedomains may vary slightly (e.g., by about 1 to 15 residues) depending onthe criteria used to define the domain. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

[0047] The invention also provides polynucleotides comprising, oralternatively consisting of, nucleic acid molecules encoding: amino acidresidues from about 1 to about 215 of SEQ ID NO:2; amino acid residuesfrom about 30 to about 215 of SEQ ID NO:2; amino acid residues fromabout 215 to about 240 of SEQ ID NO:2; amino acid residues from about240 to about 428 of SEQ ID NO:2; and amino acid residues from about 350to about 420 of SEQ ID NO:2. In this context “about” includes theparticularly recited value and values larger or smaller by several (5,4, 3, 2, or 1) amino acids. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

[0048] Preferred nucleic acid fragments of the present invention furtherinclude nucleic acid molecules encoding epitope-bearing portions of theDR3-V1 protein. In particular, such nucleic acid fragments of thepresent invention include nucleic acid molecules encoding: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 1 to about 22 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 33 to about56 in SEQ ID NO:2; a polypeptide comprising, or alternatively consistingof, amino acid residues from about 59 to about 82 in SEQ ID NO:2; apolypeptide comprising, or alternatively consisting of, amino acidresidues from about 95 to about 112 in SEQ ID NO:2; a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 122 to about 133 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 161 to about177 in SEQ ID NO:2; a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 179 to about 190 in SEQ IDNO:2; and a polypeptide comprising, or alternatively consisting of,amino acid residues from about 196 to about 205 in SEQ ID NO:2. In thiscontext “about” includes the particularly recited value and valueslarger or smaller by several (5, 4, 3, 2, or 1) amino acids. Theinventors have determined that the above polypeptide fragments areantigenic regions of the DR3-V1 protein. Methods for determining othersuch epitope-bearing portions of the DR3-V1 protein are described indetail below. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0049] Preferred nucleic acid fragments of the present invention alsoinclude nucleic acid molecules encoding epitope-bearing portions of theDR3 protein. In particular, such nucleic acid fragments of the presentinvention include nucleic acid molecules encoding the correspondingregions to those epitope-bearing regions of the DR3-V1 protein disclosedabove. Methods for determining other such epitope-bearing portions ofthe DR3 protein are described in detail below.

[0050] In another aspect, the invention provides an isolated nucleicacid molecule comprising, or alternatively consisting of, apolynucleotide which hybridizes under stringent hybridization conditionsto a portion of the polynucleotide in a nucleic acid molecule of theinvention described above, for instance, the complement of apolynucleotide fragment described herein, or the cDNA plasmids containedin ATCC Deposit 97456 or ATCC Deposit 97757. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising, or alternatively consisting of: 50% formamide, 5× SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1× SSC atabout 65° C.

[0051] By a polynucleotide which hybridizes to a “portion” of apolynucleotide is intended a polynucleotide (either DNA or RNA)hybridizing to at least about 15 nucleotides (nt), and more preferablyat least about 20 nt, still more preferably at least about 30 nt, andeven more preferably about 30-70 nt of the reference polynucleotide.These are useful as diagnostic probes and primers as discussed above andin more detail below. In this context “about” includes the particularlyrecited value and values larger or smaller by several (5, 4, 3, 2, or 1)nucleotides.

[0052] By a portion of a polynucleotide of “at least 20 nt in length,”for example, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNAs or the nucleotide sequence as shown in SEQ ID NO:1 or SEQ IDNO:3).

[0053] Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the DR3-V1 cDNA shownin SEQ ID NO:1), or to a complementary stretch of T (or U) resides,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated from an oligo-dT primed cDNAlibrary).

[0054] As indicated, nucleic acid molecules of the present inventionwhich encode the DR3-V1 or DR3 polypeptide may include, but are notlimited to the coding sequence for the mature polypeptide, by itself;the coding sequence for the mature polypeptide and additional sequences,such as those encoding a leader or secretary sequence, such as a pre-,or pro- or prepro-protein sequence; the coding sequence of the maturepolypeptide, with or without the aforementioned additional codingsequences, together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing—including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; additionalcoding sequence which codes for additional amino acids, such as thosewhich provide additional functionalities. Thus, for instance, thepolypeptide may be fused to a marker sequence, such as a peptide, whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag corresponds to an epitopederived of influenza hemagglutinin protein, which has been described byWilson et al., Cell 37:767 (1984), for instance.

[0055] The present invention further relates to variants of the nucleicacid molecules of the present invention, which encode for fragments,analogs or derivatives of the DR3-V1 or DR3 polypeptide. Variants mayoccur naturally, such as an allelic variant. By an “allelic variant” isintended one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985). Non-naturally occurring variants may beproduced using art-known mutagenesis techniques.

[0056] Such variants include those produced by nucleotide substitutions,deletions, or additions which may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions, or additions.

[0057] Further embodiments of the invention include isolated nucleicacid molecules that are at least 80% identical, and more preferably atleast 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical, to (a) anucleotide sequence encoding the full-length DR3-V1 polypeptide havingthe complete amino acid sequence in SEQ ID NO:2, including the predictedleader sequence; (b) nucleotide sequence encoding the full-length DR3polypeptide having the complete amino acid sequence in SEQ ID NO:4,including the predicted leader sequence; (c) a nucleotide sequenceencoding the mature DR3-V1 polypeptide (full-length polypeptide with theleader removed) having the amino acid sequence at positions about 36 toabout 428 in FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequence encodingthe full-length DR3-V1 polypeptide having the complete amino acidsequence including the leader encoded by the cDNA contained in ATCCDeposit No. 97456; (e) a nucleotide sequence encoding the full-lengthDR3 polypeptide having the complete amino acid sequence including theleader encoded by the cDNA contained in ATCC Deposit No. 97757; (f) anucleotide sequence encoding the mature DR3-V1 polypeptide having theamino acid sequence encoded by the cDNA contained in ATCC Deposit No.97456; (g) a nucleotide sequence encoding the mature DR3-V1 polypeptidehaving the amino acid sequence encoded by the cDNA contained in ATCCDeposit No.97757; (h) a nucleotide sequence that encodes the DR3extracellular domain; (i) a nucleotide sequence that encodes the DR3transmembrane domain; (j) a nucleotide sequence that encodes the DR3intracellular domain; (k) a nucleotide sequence that encodes the DR3death domain; or (l) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j)or (k) above. In this context “about” includes the particularly recitedvalue and values larger or smaller by several (5, 4, 3, 2, or 1) aminoacids. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0058] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aDR3-V1 or DR3 polypeptide is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five mismatches per each100 nucleotides of the reference nucleotide sequence encoding DR3-V1 orDR3. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These mismatches of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among nucleotides in the reference sequence or inone or more contiguous groups within the reference sequence. Thereference (query) sequence may be the entire DR3-V1 or DR3 encodingnucleotide sequence shown respectively in SEQ ID NO:2 and SEQ ID NO:4 orany DR3-V1 or DR3 polynucleotide fragment (e.g., a polynucleotideencoding the amino acid sequence of-any of the DR3-V1 or DR3 N- and/orC-terminal deletions described herein), variant, derivative or analog,as described herein.

[0059] As a practical matter, whether any particular nucleic acidmolecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to, for instance, the encoding nucleotide sequence shown inSEQ ID NO:2 or SEQ ID NO:4, or to the nucleotide sequence of thedeposited cDNAs, can be determined conventionally using known computerprograms such as the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981), to find the best segment of homologybetween two sequences. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referencenucleotide sequence and that gaps in homology of up to 5% of the totalnumber of nucleotides in the reference sequence are allowed.

[0060] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example; a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

[0061] The present application is directed to nucleic acid molecules atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or to thenucleic acid sequence of the deposited cDNAs, irrespective of whetherthey encode a polypeptide having DR3 functional activity. The presentapplication is also directed to nucleic acid molecules at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequences disclosed herein, (e.g., nucleic acid sequences encoding apolypeptide having the amino acid sequence of an N- and/or C-terminaldeletion disclosed herein, such as, for example, a nucleic acid moleculeencoding amino acids 30 to 200, 30 to 215, 215 to 240, 240 to 428, 350to 420, or 2 to 428 of SEQ ID NO:2), irrespective of whether they encodea polypeptide having DR3 functional activity. This is because even wherea particular nucleic acid molecule does not encode a polypeptide havingDR3 functional activity, one of skill in the art would still know how touse the nucleic acid molecule, for instance, as a hybridization probe ora polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving DR3 functional activity include, inter alia, (1) isolating theDR3 gene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the DR3-V1 or DR3 gene, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988); and (3) Northern Blot analysis for detectingDR3-V1 or DR3 mRNA expression in specific tissues.

[0062] Preferred, however, are nucleic acid molecules having sequencesat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequence shown in SEQ ID NO:1, SEQ ID NO:3 or to thenucleic acid sequence of the deposited cDNAs which do, in fact, encode apolypeptide having DR3 functional activity. By “a polypeptide having DR3functional activity” is intended polypeptides exhibiting activitysimilar, but not necessarily identical, to an activity of the DR3proteins of the invention (either the full-length protein or,preferably, the mature protein), as measured in a particular biologicalassay. For example, a DR3-V1 or DR3 functional activity can routinely bemeasured by determining the ability of a DR3-V1 or DR3 polypeptide tobind a DR3-V1 or DR3 ligand (e.g., TNF-γ-β, NF-kB, TRADD). Further, DR3functional activity can be measured using the cell death assaysperformed essentially as previously described (A. M. Chinnaiyan et al.,Cell 81: 505-12 (1995); M. P. Boldin et al., J Biol Chem 270: 7795-8(1995); F. C. Kischkel et al., EMBO 14: 5579-5588 (1995); A. M.Chinnaiyan, et al., J Biol Chem 271: 4961-4965 (1996)), and as set forthin Example 6, below. In MCF7 cells, plasmids encoding full-length DR3 ora candidate death domain containing receptors are co-transfected withthe pLantern reporter construct encoding green fluorescent protein.Nuclei of cells transfected with DR3 will exhibit apoptotic morphologyas assessed by DAPI staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzioet al., Cell 85: 817-827 (1996); M. P. Boldin et al., Cell 85: 803-815(1996); M. Tewari et al., J Biol Chem 270: 3255-60 (1995)), DR3-inducedapoptosis is blocked by the inhibitors of ICE-like proteases, CrmA andz-VAD-fmk. In addition, apoptosis induced by DR3 is also blocked bydominant negative versions of FADD (FADD-DN) or FLICE(FLICE-DN/MACHa1C360S).

[0063] The functional activity of DR3 polypeptides, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

[0064] For example, in one embodiment where one is assaying for theability to bind or compete with full-length polypeptide for binding toanti-DR3 antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

[0065] In another embodiment, where a ligand is identified (e.g.,TNF-γ-β (International Publication No. WO 00/08139, the entiredisclosure of which is incorporated herein by reference)), or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E. et al., 1995, Microbiol.Rev. 59:94-123. In another embodiment, physiological correlates ofbinding to its substrates (signal transduction) can be assayed.

[0066] Other methods will be known to the skilled artisan and are withinthe scope of the invention.

[0067] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 80%, 85%, 90%,92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence ofthe deposited cDNAs, the nucleic acid sequence shown in SEQ ID NO:2 orSEQ ID NO:4, or fragments thereof, will encode polypeptides “having DR3functional activity.” In fact, since degenerate variants of any of thesenucleotide sequences all encode the same polypeptide, in many instances,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide having DR3functional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

[0068] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that proteins are surprisingly tolerant of amino acidsubstitutions.

[0069] Polynucleotide Assays

[0070] This invention is also related to the use of the DR3-V1 or DR3polynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of a mutated form of DR3-V1or DR3 associated with a dysfunction will provide a diagnostic tool thatcan add or define a diagnosis of a disease or susceptibility to adisease which results from under-expression over-expression or alteredexpression of DR3-V1 or DR3 or a soluble form thereof, such as, forexample, tumors or autoimmune disease.

[0071] Individuals carrying mutations in the DR3-V1 or DR3 gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR prior to analysis. (Saiki et al., Nature 324:163-166 (1986)).RNA or cDNA may also be used in the same ways. As an example, PCRprimers complementary to the nucleic acid encoding DR3-V1 or DR3 can beused to identify and analyze DR3-V1 or DR3 expression and mutations. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledDR3-V1 or DR3 RNA or alternatively, radiolabeled DR3-V1 or DR3 antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0072] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotide or by automatic sequencing procedures withfluorescent-tags.

[0073] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science 230:1242 (1985)).

[0074] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.USA 85: 4397-4401 (1985)).

[0075] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”)) and Southernblotting of genomic DNA.

[0076] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0077] Chromosome Assays

[0078] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of DNAs to chromosomes according to the presentinvention is an important first step in correlating those sequences withgenes associated with disease.

[0079] In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a DR3-V1 or a DR3 gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAis then used for in situ chromosome mapping using well known techniquesfor this purpose.

[0080] In addition, sequences can be mapped to chromosomes by preparingPCR primers (preferably 15-25 bp) from the cDNA. Computer analysis ofthe 3′ untranslated region of the gene is used to rapidly select primersthat do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers are then used forPCR screening of somatic cell hybrids containing individual humanchromosomes.

[0081] Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60. For a review of this technique, see Verma et al.,Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, NewYork (1988).

[0082] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on line throughJohns Hopkins University, Welch Medical Library. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0083] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0084] Vectors and Host Cells

[0085] The present invention also relates to vectors which include DNAmolecules of the present invention, host cells which are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques.

[0086] Host cells can be genetically engineered to incorporate nucleicacid molecules and express polypeptides of the present invention. Thepolynucleotides may be introduced alone or with other polynucleotides.Such other polynucleotides may be introduced independently,co-introduced or introduced joined to the polynucleotides of theinvention.

[0087] In accordance with this aspect of the invention the vector maybe, for example, a plasmid vector, a single or double-stranded phagevector, a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides, preferably DNA,by well known techniques for introducing DNA and RNA into cells. Viralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

[0088] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector, or supplied by the vectoritself upon introduction into the host.

[0089] A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors, e.g., vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids, all maybe used for expression in accordance with this aspect of the presentinvention. Generally, any vector suitable to maintain, propagate orexpress polynucleotides to express a polypeptide in a host may be usedfor expression in this regard.

[0090] The DNA sequence in the expression vector is operatively linkedto appropriate expression control sequence(s), including, for instance,a promoter to direct mRNA transcription. Representatives of suchpromoters include the phage lambda PL promoter, the E. coli lac, trp andtac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs, to name just a few of the well-known promoters. Ingeneral, expression constructs will contain sites for transcription,initiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon (UAA, UGA orUAG) appropriately positioned at the end of the polypeptide to betranslated.

[0091] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, such regions willoperate by controlling transcription, such as repressor binding sitesand enhancers, among others.

[0092] Vectors for propagation and expression generally will includeselectable markers. Such markers also may be suitable for amplificationor the vectors may contain additional markers for this purpose. In thisregard, the expression vectors preferably contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells. Preferred markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, and tetracycline orampicillin resistance genes for culturing E. coli and other bacteria.

[0093] The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS, and Bowes melanoma cells; and plant cells. Hosts for agreat variety of expression constructs are well known, and those ofskill will be enabled by the present disclosure readily to select a hostfor expressing a polypeptides in accordance with this aspect of thepresent invention. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

[0094] Among vectors preferred for use in bacteria are pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. These vectors are listed solely by way ofillustration of the many commercially available and well known vectorsavailable to those of skill in the art.

[0095] Selection of appropriate vectors and promoters for expression ina host cell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host are routine skills in the art.

[0096] The present invention also relates to host cells containing theabove-described constructs discussed above. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell.

[0097] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods in MolecularBiology (1986).

[0098] In addition to encompassing host cells containing the vectorconstructs discussed herein, the invention also encompasses primary,secondary, and immortalized host cells of vertebrate origin,particularly mammalian origin, that have been engineered to delete orreplace endogenous genetic material (e.g., the DR3 coding sequence),and/or to include genetic material (e.g., heterologous polynucleotidesequences) that is operably associated with DR3-V1 or DR3polynucleotides of the invention, and which activates, alters, and/oramplifies endogenous DR3-V1 or DR3 polynucleotides. For example,techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous DR3-V1 or DR3 polynucleotide sequences via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication Number WO 96/29411, published Sep. 26, 1996;International Publication Number WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

[0099] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL-5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16:9459-9471 (1995).

[0100] The DR3 and DR3-V1 polypeptides can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and/or purification.

[0101] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0102] DR3-V1 or DR3 polynucleotides and polypeptides may be used inaccordance with the present invention for a variety of applications,particularly those that make use of the chemical and biologicalproperties of DR3. Among these are applications in treatment and/orprevention of tumors, resistance to parasites, bacteria and viruses, toinduce proliferation of T-cells, endothelial cells and certainhematopoietic cells, to treat and/or prevent restenosis, graft vs. hostdisease, to regulate anti-viral responses and to prevent certainautoimmune diseases after stimulation of DR3 by an agonist. Additionalapplications relate to the prognosis, diagnosis, prevention and/ortreatment of disorders of cells, tissues and organisms. These aspects ofthe invention are discussed further below.

[0103] DR3 Polypeptides and Fragments

[0104] The invention further provides an isolated DR3-V1 or DR3polypeptide having the amino acid sequence shown in SEQ ID NO:2 and SEQID NO:4, respectively, or a fragment thereof. It will be recognized inthe art that some amino acid sequence of DR3-V1 or DR3 can be variedwithout significant effect of the structure or function of the protein.If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity. Such areas will usually comprise residues which makeup the ligand binding site or the death domain, or which form tertiarystructures which affect these domains.

[0105] Thus, the invention further includes variations of the DR3-V1 orDR3 protein which show substantial DR3 functional activity or whichinclude regions of DR3-V1 or DR3 such as the protein fragments discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions. As indicated above, guidance concerning whichamino acid changes are likely to be phenotypically silent can be foundin J. U. Bowie et al., Science 247:1306-1310 (1990).

[0106] Of particular interest are substitutions of charged amino acidswith another charged amino acid and with neutral or negatively chargedamino acids. The latter results in proteins with reduced positive chargeto improve the characteristics of the DR3-V1 or DR3 protein. Theprevention of aggregation is highly desirable. Aggregation of proteinsnot only results in a loss of activity but can also be problematic whenpreparing pharmaceutical formulations, because they can be immunogenic.(Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al.,Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic DrugCarrier Systems 10:307-377 (1993)).

[0107] The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, theDR3-V1 or DR3 receptor of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation.

[0108] As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1). TABLE 1Conservative Amino Acid Substitutions. Aromatic Phenylalanine TryptophanTyrosine Hydrophobic Leucine Isoleucine Valine Polar GlutamineAsparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid GlutamicAcid Small Alanine Serine Threonine Methionine Glycine

[0109] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabove. Generally speaking, the number of substitutions for any givenDR3-V1 or DR3 polypeptide will not be more than 50, 40, 30, 25, 20, 15,10, 5 or 3.

[0110] Amino acids in the DR3-V1 or DR3 protein of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro, or in vitroproliferative activity. Sites that are critical for ligand-receptorbinding can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al.Science 255:306-312 (1992)).

[0111] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Arecombinantly produced version of the DR3-V1 or DR3 polypeptide issubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988).

[0112] The polypeptides of the present invention also include thepolypeptide encoded by the deposited cDNAs including the leader, themature polypeptide encoded by the deposited the cDNAs minus the leader(i.e., the mature protein), the polypeptide of SEQ ID NO:2 or SEQ IDNO:4 including the leader, the polypeptide of SEQ ID NO:2 or SEQ ID NO:4minus the leader, the extracellular domain, the transmembrane domain,the intracellular domain, soluble polypeptides comprising, oralternatively consisting of, all or part of the extracellular andintracellular domains but lacking the transmembrane domain as well aspolypeptides which are at least 80% identical, more preferably at least80% or 85% identical, still more preferably at least 90%, 92%, 95%, 96%,97%, 98% or 99% identical to the polypeptide encoded by the depositedcDNAs, to the polypeptide of SEQ ID NO:2 or SEQ ID NO:4, and alsoinclude portions of such polypeptides with at least 30 amino acids andmore preferably at least 50 amino acids. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0113] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a DR3-V1or DR3 polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of a DR3-V1 or DR3. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0114] As a practical matter, whether any particular polypeptide is atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4,the amino acid sequence encoded by the deposited cDNAs, or fragmentsthereof, can be determined conventionally using known computer programssuch as the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). When using Bestfit or any othersequence alignment program to determine whether a particular sequenceis, for instance, 95% identical to a reference sequence according to thepresent invention, the parameters are set, of course, such that thepercentage of identity is calculated over the full length of thereference amino acid sequence and that gaps in homology of up to 5% ofthe total number of amino acid residues in the reference sequence areallowed.

[0115] In a specific embodiment, the identity between a reference(query) sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, is determinedusing the FASTDB computer program based on the algorithm of Brutlag etal. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection is made to the results to take into consideration the factthat the PASTDB program does not account for N- and C-terminaltruncations of the subject sequence when calculating global percentidentity. For subject sequences truncated at the N- and C-termini,relative to the query sequence, the percent identity is corrected bycalculating the number of residues of the query sequence that are N- andC-terminal of the subject sequence, which are not matched/aligned with acorresponding subject residue, as a percent of the total bases of thequery sequence. A determination of whether a residue is matched/alignedis determined by results of the FASTDB sequence alignment. Thispercentage is then subtracted from the percent identity, calculated bythe above FASTDB program using the specified parameters, to arrive at afinal percent identity score. This final percent identity score is whatis used for the purposes of this embodiment. Only residues to the N- andC-termini of the subject sequence, which are not matched/aligned withthe query sequence, are considered for the purposes of manuallyadjusting the percent identity score. That is, only query residuepositions outside the farthest N- and C-terminal residues of the subjectsequence. For example, a 90 amino acid residue subject sequence isaligned with a 100 residue query sequence to determine percent identity.The deletion occurs at the N-terminus of the subject sequence andtherefore, the FASTDB alignment does not show a matching/alignment ofthe first 10 residues at the N-terminus. The 10 unpaired residuesrepresent 10% of the sequence (number of residues at the N- andC-termini not matched/total number of residues in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched thefinal percent identity would be 90%. In another example, a 90 residuesubject sequence is compared with a 100 residue query sequence. Thistime the deletions are internal deletions so there are no residues atthe N- or C-termini of the subject sequence which are notmatched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected for. No other manualcorrections are made for the purposes of this embodiment.

[0116] The present inventors have discovered that the DR3-V1 polypeptideis a 428 residue protein exhibiting three main structural domains.First, the ligand binding domain was identified within amino acidresidues from about 36 to about 212 in SEQ ID NO:2. Second, thetransmembrane domain was identified within amino acid residues fromabout 213 to about 235 in SEQ ID NO:2. Third, the intracellular domainwas identified within amino acid residues from about 236 to about 428 inSEQ ID NO:2. Importantly, the intracellular domain includes a deathdomain at amino acid residues from about 353 to about 419. Furtherpreferred fragments of the polypeptide shown in SEQ ID NO:2 include themature protein from amino acid residues about 36 to about 428 andsoluble polypeptides comprising, or alternatively consisting of, all orpart of the extracellular and intracellular domains but lacking thetransmembrane domain. In this context “about” includes the particularlyrecited value and values larger or smaller by several (5, 4, 3, 2, or 1)amino acids. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0117] The invention also provides polypeptides comprising, oralternatively consisting of, one, two, three, four, five or more aminoacid sequences selected from the group consisting of amino acid residuesfrom about 1 to about 215 of SEQ ID NO:2; amino acid residues from about30 to about 215 of SEQ ID NO:2; amino acid residues from about 215 toabout 240 of SEQ ID NO:2; amino acid residues from about 240 to about428 of SEQ ID NO:2; and amino acid residues from about 350 to about 420of SEQ ID NO:2. In this context “about” includes the particularlyrecited value and values larger or smaller by several (5, 4, 3, 2, or 1)amino acids. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0118] The present inventors have also discovered that the DR3polypeptide is a 417 residue protein exhibiting three main structuraldomains. First, the ligand binding domain was identified within aminoacid residues from about 25 to about 201 in SEQ ID NO:4. Second, thetransmembrane domain was identified within amino acid residues fromabout 202 to about 224 in SEQ ID NO:4. Third, the intracellular domainwas identified within amino acid residues from about 225 to about 417 inSEQ ID NO:4. Importantly, the intracellular domain includes a deathdomain at amino acid residues from about 342 to about 408. Furtherpreferred fragments of the polypeptide shown in SEQ ID NO:4 include themature protein from amino acid residues about 25 to about 417 andsoluble polypeptides comprising, or alternatively consisting of, all orpart of the extracellular and intracellular domains but lacking thetransmembrane domain. In this context “about” includes the particularlyrecited value and values larger or smaller by several (5, 4, 3, 2, or 1)amino acids. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0119] As one of skill in the art will recognize, the full lengthpolypeptides encoded by the DR3-V1 and DR3 cDNA differ only in the aminoacid sequence of the leader peptide. The first 24 amino acids of thepolypeptide shown in SEQ ID NO:2 are replaced by the first 13 aminoacids shown in SEQ ID NO:4 but the rest of the amino acid sequence isthe same. Thus, both the DR3-V1 cDNA and DR3 cDNA encode an identicalmature protein having the same biological activity.

[0120] Thus, the invention further provides DR3-V1 or DR3 polypeptidesencoded by the deposited cDNAs including the leader and DR3-V1 or DR3polypeptide fragments selected from the mature protein, theextracellular domain, the transmembrane domain, the intracellulardomain, and the death domain.

[0121] The polypeptides of the present invention have uses whichinclude, but are not limited to, as sources for generating antibodiesthat bind the polypeptides of the invention, and as molecular weightmarkers on SDS-PAGE gels or on molecular sieve gel filtration columnsusing methods well known to those of skill in the art.

[0122] In another aspect, the invention provides a peptide orpolypeptide comprising, or alternatively consisting of, anepitope-bearing portion of a polypeptide described herein.

[0123] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies that bind specifically to a polypeptide of the invention.See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

[0124] Antigenic epitope-bearing peptides and polypeptides of theinvention preferably contain a sequence of at least seven, morepreferably at least nine and most preferably between at least about 15to about 30 amino acids contained within the amino acid sequence of apolypeptide of the invention. In this context “about” includes theparticularly recited value and values larger or smaller by several (5,4, 3, 2, or 1) amino acids. Polynucleotides encoding these antigenicepitope-bearing peptides are also encompassed by the invention.

[0125] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate DR3-specific antibodies include: a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 1 to about 22 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, one, two, three, four, five or more aminoacid sequences selected from the group consisting of amino acid residuesfrom about 33 to about 56 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 59 to about82 in SEQ ID NO:2; a polypeptide comprising, or alternatively consistingof, amino acid residues from about 95 to about 112 in SEQ ID NO:2; apolypeptide comprising, or alternatively consisting of, amino acidresidues from about 122 to about 133 in SEQ ID NO:2; a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout 161 to about 177 in SEQ ID NO:2; a polypeptide comprising, oralternatively consisting of, amino acid residues from about 179 to about190 in SEQ ID NO:2; and a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about 196 to about 205 in SEQ IDNO:2. In this context “about” includes the particularly recited valueand values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.Polynucleotides encoding these antigenic epitope-bearing peptides arealso encompassed by the invention. In addition, antigenic polypeptidesor peptides include polypeptides comprising, or alternatively consistingof, the amino acid residues that are the corresponding residues to thosepolypeptides of DR3-V1 disclosed above. As indicated above, theinventors have determined that the above polypeptide fragments areantigenic regions of the DR3-V1 and DR3 protein.

[0126] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means. R. A. Houghten, “Generalmethod for the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

[0127] As one of skill in the art will appreciate, DR3-V1 or DR3polypeptides of the present invention, and the epitope-bearing fragmentsthereof, described above, can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric DR3-V1 or DR3 protein orprotein fragment alone (Fountoulakis et al., J Biochem 270:3958-3964(1995)).

[0128] The present invention thus encompasses polypeptides comprising,or alternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or an epitope of thepolypeptide sequence encoded by a polynucleotide sequence contained inthe plasmid deposited as ATCC Deposit No. 97456 or 97757 or encoded by apolynucleotide that hybridizes to the complement of the sequence of SEQID NO:1 or SEQ ID NO:3 or contained in the plasmid deposited as ATCCDeposit No. 97456 or 97757 under stringent hybridization conditions orlower stringency hybridization conditions as defined supra. The presentinvention further encompasses polynucleotide sequences encoding anepitope of a polypeptide sequence of the invention (such as, forexample, the sequence disclosed in SEQ ID NO:1 or SEQ ID NO:3),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra. Polynucleotides encoding these antigenic epitope-bearingpeptides are also encompassed by the invention.

[0129] The term “epitopes,” as used herein, refers to portions of apolypeptide having antigenic or immunogenic activity in an animal,preferably a mammal, and most preferably in a human. In a preferredembodiment, the present invention encompasses a polypeptide comprisingan epitope, as well as the polynucleotide encoding this polypeptide. An“immunogenic epitope,” as used herein, is defined as a portion of aprotein that elicits an antibody response in an animal, as determined byany method known in the art, for example, by the methods for generatingantibodies described infra. (See, for example, Geysen et al., Proc.Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,”as used herein, is defined as a portion of a protein to which anantibody can immunospecifically bind its antigen as determined by anymethod well known in the art, for example, by the immunoassays describedherein. Immunospecific binding excludes non-specific binding but doesnot necessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

[0130] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, J. G. Sutcliffeet al., “Antibodies that react with predetermined sites on proteins,”Science 219:660-666 (1983). Peptides capable of elicitingprotein-reactive sera are frequently represented in the primary sequenceof a protein, can be characterized by a set of simple chemical rules,and are confined neither to immunodominant regions of intact proteins(i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

[0131] Fragments that function as epitopes may be produced by anyconventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).

[0132] In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)).

[0133] Similarly, immunogenic epitopes can be used, for example, toinduce antibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985)). The polypeptides comprising one or moreimmunogenic epitopes may be presented for eliciting an antibody responsetogether with a carrier protein, such as an albumin, to an animal system(such as, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

[0134] Epitope-bearing polypeptides of the present invention may be usedto induce antibodies according to methods well known in the artincluding, but not limited to, in vivo immunization, in vitroimmunization, and phage display methods. See, e.g., Sutcliffe et al.,supra; Wilson et al., supra, and Bittle et al., supra. If in vivoimmunization is used, animals may be immunized with free peptide;however, anti-peptide antibody titer may be boosted by coupling thepeptide to a macromolecular carrier, such as keyhole limpet hemacyanin(KLH) or tetanus toxoid. For instance, peptides containing cysteineresidues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed; for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

[0135] As one of skill in the art will appreciate, and as discussedabove, the polypeptides of the present invention comprising animmunogenic or antigenic epitope can be fused to other polypeptidesequences. For example, the polypeptides of the present invention may befused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM),or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins. See,e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgGFusion proteins that have a disulfide-linked dimeric structure due tothe IgG portion disulfide bonds have also been found to be moreefficient in binding and neutralizing other molecules than monomericpolypeptides or fragments thereof alone. See, e.g., Fountoulakis et al.,J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the aboveepitopes can also be recombined with a gene of interest as an epitopetag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detectionand purification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion.protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

[0136] Polypeptides of the invention (including antibodies of theinvention, see below) may also be fused to albumin (including but notlimited to recombinant human serum albumin (see, e.g., U.S. Pat. No.5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No.5,766,883, issued Jun. 16, 1998, herein incorporated by reference intheir entirety)), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides (including antibodies) of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094, which is hereinincorporated by reference in its entirety). In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883, herein incorporated byreference in its entirety. Polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) may be fused toeither the N- or C-terminal end of the heterologous protein (e.g.,immunoglobulin Fc polypeptide or human serum albumin polypeptide). Suchhuman serum albumin DR3 and/or DR3-V1 fusion proteins may be usedtherapeutically in accordance with the invention, in the same manner as,for example, the DR3 and/or DR3-V1 Fc fusion proteins described herein.

[0137] Additional fusion proteins of the invention may be generatedthrough the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr.Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol.16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); andLorenzo and Blasco, BioTechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference in itsentirety). In one embodiment, alteration of polynucleotidescorresponding to SEQ ID NO:1 or SEQ ID NO:3 and the polypeptides encodedby these polynucleotides may be achieved by DNA shuffling. DNA shufflinginvolves the assembly of two or more DNA segments by homologous orsite-specific recombination to generate variation in the polynucleotidesequence. In another embodiment, polynucleotides of the invention, orthe encoded polypeptides, may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. In another embodiment, one or morecomponents, motifs, sections, parts, domains, fragments, etc., of apolynucleotide coding a polypeptide of the invention may be recombinedwith one or more components, motifs, sections, parts, domains,fragments, etc. of one or more heterologous molecules.

[0138] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindDR3-V1 ligand) may still be retained. For example, the ability ofshortened DR3-V1 muteins to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptides generallywill be retained when less than the majority of the residues of thecomplete or mature polypeptide are removed from the N-terminus. Whethera particular polypeptide lacking N-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an DR3-V1 mutein with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixDR3-V1 amino acid residues may often evoke an immune response.

[0139] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of theDR3-V1 amino acid sequence shown in SEQ ID NO:2, up to the arginineresidue at position number 423 and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues n1-428 of SEQ ID NO:2, where n1 is an integer from 2 to 423corresponding to the position of the amino acid residue in SEQ ID NO:2.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0140] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues E-2 to P-428; E-3 to P-428; T-4 to P-428; Q-5 toP-428; Q-6 to P-428; G-7 to P-428; E-8 to P-428; A-9 to P-428; P-10 toP-428; R-11 to P-428; G-12 to P428; Q-13 to P-428; L-14 to P-428; R-15to P-428; G-16 to P-428; E-17 to P-428; S-18 to P-428; A-19 to P-428;A-20 to P-428; P-21 to P-428; V-22 to P-428; P-23 to P-428; Q-24 toP-428; A-25 to P-428; L-26 to P-428; L-27 to P-428; L-28 to P-428; V-29to P-428; L-30 to P-428; L-31 to P-428; G-32 to P-428; A-33 to P-428;R-34 to P-428; A-35 to P-428; Q-36 to P-428; G-37 to P-428; G-38 toP-428; T-39 to P-428; R-40 to P-428; S-41 to P-428; P-42 to P-428; R-43to P-428; C-44 to P-428; D-45 to P-428; C-46 to P-428; A-47 to P-428;G-48 to P-428; D-49 to P-428; F-50 to P-428; H-51 to P-428; K-52 toP-428; K-53 to P-428; I-54 to P-428; G-55 to P-428; L-56 to P-428; F-57to P-428; C-58 to P-428; C-59 to P-428; R-60 to P-428; G-61 to P-428;C-62 to P-428; P-63 to P-428; A-64 to P-428; G-65 to P-428; H-66 toP-428; Y-67 to P-428; L-68 to P-428; K-69 to P-428; A-70 to P-428; P-71to P-428; C-72 to P-428; T-73 to P-428; E-74 to P-428; P-75 to P-428;C-76 to P-428; G-77 to P-428; N-78 to P-428; S-79 to P-428; T-80 toP-428; C-81 to P-428; L-82 to P-428; V-83 to P-428; C-84 to P-428; P-85to P-428; Q-86 to P-428; D-87 to P-428; T-88 to P-428; F-89 to P-428;L-90 to P-428; A-91 to P-428; W-92 to P-428; E-93 to P-428; N-94 toP-428; H-95 to P-428; H-96 to P-428; N-97 to P-428; S-98 to P-428; E-99to P-428; C-100 to P-428; A-101 to P-428; R-102 to P-428; C-103 toP-428; Q-104 to P-428; A-105 to P-428; C-106 to P428; D-107 to P-428;E-108 to P-428; Q-109 to P-428; A-110 to P-428; S-111 to P-428; Q-112 toP-428; V-113 to P-428; A-114 to P-428; L-115 to P-428; E-116 to P428;N-117 to P428; C-1 18 to P-428; S-119 to P-428; A-120 to P-428; V-121 toP-428; A-122 to P-428; D-123 to P-428; T-124 to P-428; R-125 to P-428;C-126 to P-428; G-127 to P-428; C-128 to P-428; K-129 to P-428; P-130 toP-428; G-131 to P-428; W-132 to P-428; F-133 to P-428; V-134 to P-428;E-135 to P-428; C-136 to P-428; Q-137 to P-428; V-138 to P-428; S-139 toP-428; Q-140 to P-428; C-141 to P-428; V-142 to P-428; S-143 to P-428;S-144 to P-428; S-145 to P428; P-146 to P-428; F-147 to P-428; Y-148 toP-428; C-149 to P-428; Q-150 to P-428; P-151 to P-428; C-152 to P-428;L-153 to P-428; D-154 to P-428; C-155 to P-428; G-156 to P-428; A-157 toP-428; L-158 to P-428; H-159 to P-428; R-160 to P-428; H-161 to P-428;T- 162 to P-428; R-163 to P-428; L-164 to P-428; L-165 to P-428; C-166to P-428; S-167 to P-428; R-168 to P-428; R-169 to P-428; D-170 toP-428; T-171 to P-428; D-172 to P-428; C-173 to P-428; G-174 to P-428;T-175 to P-428; C-176 to P-428; L-177 to P-428; P-178 to P-428; G-179 toP-428; F-180 to P-428; Y-181 to P-428; E-182 to P-428; H-183 to P-428;G-184 to P-428; D-185 to P-428; G-186 to P-428; C-187 to P-428; V-1 88to P-428; S-1 89 to P-428; C-190 to P-428; P-191 to P-428; T-192 toP-428; S-193 to P-428; T-194 to P-428; L-195 to P-428; G-196 to P-428;S-197 to P-428; C-198 to P-428; P-199 to P-428; E-200 to P-428; R-201 toP-428; C-202 to P-428; A-203, to P-428; A-204 to P-428; V-205 to P-428;C-206 to P-428; G-207 to P-428; W-208 to P-428; R-209 to P-428; Q-210 toP-428; M-211 to P-428; F-212 to P-428; W-213 to P-428; V-214 to P-428;Q-215 to P-428; V-216 to P-428; L-217 to P-428; L-218 to P-428; A-219 toP-428; G-220 to P-428; L-221 to P-428; V-222 to P428; V-223 to P-428;P-224 to P-428; L-225 to P-428; L-226 to P-428; L-227 to P-428; G-228 toP-428; A-229 to P428; T-230 to P-428; L-231 to P-428; T-232 to P-428;Y-233 to P-428; T-234 to P-428; Y-235 to P-428; R-236 to P-428; H-237 toP-428; C-238 to P-428; W-239 to P-428; P-240 to P-428; H-241 to P-428;K-242 to P-428; P-243 to P-428; L-244 to P-428; V-245 to P-428; T-246 toP-428; A-247 to P-428; D-248 to P-428; E-249 to P-428; A-250 to P-428;G-251 to P-428; M-252 to P-428; E-253 to P-428; A-254 to P-428; L-255 toP-428; T-256 to P-428; P-257 to P-428; P-258 to P-428; P-259 to P-428;A-260 to P-428; T-261 to P-428; H-262 to P-428; L-263 to P-428; S-264 toP428; P-265 to P428; L-266 to P-428; D-267 to P-428; S-268 to P-428;A-269 to P428; H-270 to P-428; T-271 to P-428; L-272 to P-428; L-273 toP-428; A-274 to P-428; P-275 to P-428; P-276 to P-428; D-277 to P-428;S-278 to P-428; S-279 to P-428; E-280 to P-428; K-281 to P-428; I-282 toP-428; C-283 to P-428; T-284 to P-428; V-285 to P-428; Q-286 to P-428;L-287 to P-428; V-288 to P-428; G-289 to P-428; N-290 to P-428; S-291 toP-428; W-292 to P-428; T-293 to P-428; P-294 to P-428; G-295 to P-428;Y-296 to P-428; P-297 to P-428; E-298 to P-428; T-299 to P-428; Q-300 toP-428; E-301 to P-428; A-302 to P-428; L-303 to P-428; C-304 to P-428;P-305 to P-428; Q-306 to P-428; V-307 to P-428; T-308 to P-428; W-309 toP-428; S-310 to P-428; W-311 to P-428; D-312 to P-428; Q-313 to P-428;L-314 to P-428; P-315 to P-428; S-316 to P-428; R-317 to P-428; A-318 toP-428; L-319 to P-428; G-320 to P-428; P-321 to P-428; A-322 to P-428;A-323 to P-428; A-324 to P-428; P-325 to P-428; T-326 to P-428; L-327 toP-428; S-328 to P-428; P-329 to P-428; E-330 to P-428; S-331 to P428;P-332 to P-428; A-333 to P-428; G-334 to P-428; S-335 to P-428; P-336 toP-428; A-337 to P-428; M-338 to P-428; M-339 to P-428; L-340 to P-428;Q-341 to P-428; P-342 to P-428; G-343 to P-428; P-344 to P-428; Q-345 toP-428; L-346 to P-428; Y-347 to P-428; D-348 to P-428; V-349 to P-428;M-350 to P-428; D-351 to P-428; A-352 to P-428; V-353 to P-428; P-354 toP-428; A-355 to P-428; R-356 to P-428; R-357 to P-428; W-358 to P428;K-359 to P-428; E-360 to P-428; F-361 to P-428; V-362 to P-428; R-363 toP-428; T-364 to P-428; L-365 to P-428; G-366 to P-428; L-367 to P-428;R-368 to P428; E-369 to P-428; A-370 to P-428; E-371 to P-428; 1-372 toP-428; E-373 to P-428; A-374 to P-428; V-375 to P-428; E-376 to P428;V-377 to P-428; E-378 to P428; I-379 to P428; G-380 to P-428; R-381 toP-428; F-382 to P-428; R-383 to P-428; D-384 to P-428; Q-385 to P-428;Q-386 to P-428; Y-387 to P-428; E-388 to P-428; M-389 to P-428; L-390 toP-428; K-391 to P-428; R-392 to P428; W-393 to P-428; R-394 to P-428;Q-395 to P-428; Q-396 to P-428; Q-397 to P-428; P-398 to P-428; A-399 toP-428; G-400 to P-428; L-401 to P-428; G-402 to P-428; A-403 to P-428;V-404 to P-428; Y-405 to P-428; A-406 to P-428; A-407 to P-428; L-408 toP-428; E-409 to P428; R-410 to P-428; M-411 to P-428; G-412 to P-428;L-413 to P-428; D-414 to P-428; G-415 to P-428; C-416 to P-428; V-417 toP-428; E-418 to P-428; D-419 to P-428; L-420 to P-428; R-421 to P-428;S-422 to P-428; and R-423 to P-428 of the DR3-V1 sequence shown in SEQID NO:2. The present invention is also directed to nucleic acidmolecules comprising, or alternatively consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to the polynucleotide sequences encoding the polypeptidesdescribed above. The present invention also encompasses the abovepolynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0141] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind DR3-V1 ligand) may still be retained. For example the ability ofthe shortened DR3-V1 mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an DR3-V1 mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixDR3-V1 amino acid residues may often evoke an immune response.

[0142] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the DR3-V1 polypeptide shown in SEQ ID NO:2, upto the glutamine residue at position number 6, and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues 1-ml of SEQ ID NO:2, where m1 is aninteger from 6 to 427 corresponding to the position of the amino acidresidue in SEQ ID NO:2. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

[0143] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues M-1 to G-427; M-1 to R-426; M-1 to Q-425; M-1 toL-424; M-1 to R423; M-1 to S-422; M-1 to R-421; M-1 to L-420; M-1 toD-419; M-1 to E-418; M-1 to V-417; M-1 to C-416; M-1 to G-415; M-1 toD-414; M-1 to L-413; M-1 to G-412; M-1 to M-411; M-1 to R-410; M-1 toE-409; M-1 to L-408; M-1 to A-407; M-1 to A-406; M-1 to Y-405; M-1 toV-404; M-1 to A-403; M-1 to G-402; M-1 to L-401; M-1 to G-400; M-1 toA-399; M-1 to P-398; M-1 to Q-397; M-1 to Q-396; M-1 to Q-395; M-1 toR-394; M-1 to W-393; M-1 to R-392; M-1 to K-391; M-1 to L-390; M-1 toM-389; M-1 to E-388; M-1 to Y-387; M-1 to Q-386; M-1 to Q-385; M-1 toD-384; M-1 to R-383; M-1 to F-382; M-1 to R-381; M-1 to G-380; M-1 toI-379; M-1 to E-378; M-1 to V-377; M-1 to E-376; M-1 to V-375; M-1 toA-374; M-1 to E-373; M-1 to I-372; M-1 to E-371; M-1 to A-370; M-1 toE-369; M-1 to R-368; M-1 to L-367; M-1 to G-366; M-1 to L-365; M-1 toT-364; M-1 to R-363; M-1 to V-362; M-1 to F-361; M-1 to E-360; M-1 toK-359; M-1 to W-358; M-1 to R-357; M-1 to R-356; M-1 to A-355; M-1 toP-354; M-1 to V-353; M-1 to A-352; M-1 to D-351; M-1 to M-350; M-1 toV-349; M-1 to D-348; M-1 to Y-347; M-1 to L-346; M-1 to Q-345; M-1 toP-344; M-1 to G-343; M-1 to P-342; M-1 to Q-341; M-1 to L-340; M-1 toM-339; M-1 to M-338; M-1 to A-337; M-1 to P-336; M-1 to S-335; M-1 toG-334; M-1 to A-333; M-1 to P-332; M-1 to S-331; M-1 to E-330; M-1 toP-329; M-1 to S-328; M-1 to L-327; M-1 to T-326; M-1 to P-325; M-1 toA-324; M-1 to A-323; M-1 to A-322;. M-1 to P-321; M-1 to G-320; M-1 toL-319; M-1 to A-318; M-1 to R-317; M-1 to S-316; M-1 to P-315; M-1 toL-314; M-1 to Q-313; M-1 to D-312; M-1 to W-311; M-1 to S-310; M-1 toW-309; M-1 to T-308; M-1 to V-307; M-1 to Q-306; M-1 to P-305; M1 toC-304; M-1 to L-303; M-1 to A-302; M-1 to E-301; M-1 to Q-300; M-1 toT-299; M-1 to E-298; M-1 to P-297; M-1 to Y-296; M-1 to G-295; M-1 toP-294; M-1 to T-293; M-1 to W-292; M-1 to S-291; M-1 to N-290; M-1 toG-289; M-1 to V-288; M-1 to L-287; M-1 to Q-286; M-1 to V-285; M -1 toT-284; M-1 to C-283; M-1 to I-282; M-1 to K-281; M-1 to E-280; M-1 toS-279; M-1 to S-278; M-1 to D-277; M-1 to P-276; M-1 to P-275; M-1 toA-274; M-1 to L-273; M-1 to L-272; M-1 to T-271; M-1 to H-270; M-1 toA-269; M-1 to S-268; M-1 to D-267; M-1 to L-266; M-1 to P-265; M-1 toS-264; M-1 to L-263; M-1 to H-262; M-1 to T-261; M-1 to A-260; M-1 toP-259; M-1 to P-258; M-1 to P-257; M-1 to T-256; M-1 to L-255; M-1 toA-254; M-1 to E-253; M-1 to M-252; M-1 to G-251; M-1 to A-250; M-1 toE-249; M-1 to D-248; M-1 to A-247; M-1 to T-246; M-1 to V-245; M-1 toL-244; M-1 to P-243; M-1 to K-242; M-1 to H-241; M-1 to P-240; M-1 toW-239; M-1 to C-238; M-1 to H-237; M-1 to R-236; M-1 to Y-235; M-1 toT-234; M-1 to Y-233; M-1 to T-232; M-1 to L-231; M-1 to T-230; M-1 toA-229; M-1 to G-228; M-1 to L-227; M-1 to L-226; M-1 to L-225; M-1 toP-224; M-1 to V-223; M-1 to V-222; M-1 to L-221; M-1 to G-220; M-1 toA-219; M-1 to L-218; M-1 to L-217; M-1 to V-216; M-1 to Q-215; M-1 toV-214; M-1 to W-213; M-1 to F-212; M-1 to M-211; M-1 to Q-210; M-1 toR-209; M-1 to W-208; M-1 to G-207; M-1 to C-206; M-1 to V-205; M-1 toA-204; M-1 to A-203; M-1 to C-202; M-1 to R-201; M-1 to E-200; M-1 toP-199; M-1 to C-198; M-1 to S-197; M-1 to G-196; M-1 to L-195; M-1 toT-194; M-1 to S-193; M-1 to T-192; M-1 to P-191; M-1 to C-190; M-1 toS-189; M-1 to V-188; M-1 to C-187; M-1 to G-186; M-1 to D-185; M-1 toG-184; M-1 to H-183; M-1 to E-182; M-1 to Y-181; M-1 to F-180; M-1 toG-179; M-1 to P-178; M-1 to L-177; M-1 to C-176; M-1 to T-175; M-1 toG-174; M-1 to C-173; M-1 to D-172; M-1 to T-171; M -1 to D-170; M-1 toR-169; M-1 to R-168; M-1 to S-167; M-1 to C-166; M-1 to L-165; M-1 toL-164; M-1 to R-163; M-1 to T-162; M-1 to H-161; M-1 to R-160; M-1 toH-159; M-1 to L-158; M-1 to A-157; M-1 to G-156; M-1 to C-155; M-1 toD-154; M-1 to L-153; M-1 to C-152; M-1 to P-151; M-1 to Q-150; M-1 toC-149; M-1 to Y-148; M-1 to F-147; M-1 to P-146; M-1 to S-145; M -1 toS-144; M-1 to S-143; M-1 to V-142; M-1 to C-141; M-1 to Q-140; M-1 toS-139; M-1 to V-138; M-1 to Q-137; M-1 to C-136; M-1 to E-135; M-1to.V-134; M-1 to F-133; M-1 to W-132; M-1 to G-131; M-1 to P-130; M-1 toK-129; M-1 to C-128; M-1 to G-127; M-1 to C-126; M-1 to R-125; M-1 toT-124; M-1 to D-123; M-1 to A-122; M-1 to V-121; M-1 to A-120; M-1 toS-119; M-1 to C-118; M-1 to N-117; M-1 to E-116; M-1 to L-115; M-1 toA-114; M-1 to V-113; M-1 to Q-112; M-1 to S-11; M-1 to A-110; M-1 toQ-109; M-1 to E-108; M-1 to D-107; M-1 to C-106; M-1 to A-105; M-1 toQ-104; M-1 to C-103; M-1 to R-102; M-1 to A-101; M-1 to C-100; M-1 toE-99; M-1 to S-98; M-1 to N-97; M-1 to H-96; M-1 to H-95; M-1 to N-94;M-1 to E-93; M-1 to W-92; M-1 to A-91; M-1 to L-90; M-1 to F-89; M-1 toT-88; M-1 to D-87; M-1 to Q-86; M-1 to P-85; M-1 to C-84; M-1 to V-83;M-1 to L-82; M-1 to C-81; M-1 to T-80; M-1 to S-79; M-1 to N-78; M-1 toG-77; M-1 to C-76; M-1 to P-75; M-1 to E-74; M-1 to T-73; M-1 to C-72;M-1 to P-71; M-1 to A-70; M-1 to K-69; M-1 to L-68; M-1 to Y-67; M-1 toH-66; M-1 to G-65; M-1 to A-64; M-1 to P-63; M-1 to C-62; M-1 to G-61;M-1 to R-60; M-1 to C-59; M-1 to C-58; M-1 to F-57; M-1 to L-56; M-1 toG-55; M-1 to I-54; M-1 to K-53; M-1 to K-52; M-1 to H-5 1; M-1 to F-50;M-1 to D-49; M-1 to G-48; M-1 to A-47; M-1 to C-46; M-1 to D-45; M-1 toC-44; M-1 to R-43; M-1 to P-42; M-1 to S-41; M-1 to R-40; M-1 to T-39;M-1 to G-38; M-1 to G-37; M-1 to Q-36; M-1 to A-35; M-1 to R-34; M-1 toA-33; M-1 to G-32; M-1 to L-31; M-1 to L-30; M-1 to V-29; M-1 to L-28;M-1 to L-27; M-1 to L-26; M-1 to A-25; M-1 to Q-24; M-1 to P-23; M-1 toV-22; M-1 to P-21; M-1 to A-20; M-1 to A-19; M-1 to S-18; M-1 to E-17;M-1 to G-16; M-1 to R-15; M-1 to L-14; M-1 to Q-13; M-1 to G-12; M-1 toR-1 1; M-1 to P-10; M-1 to A-9; M-1 to E-8; M-1 to G-7; and M-1 to Q-6of the sequence of the DR3-V1 sequence shown in SEQ ID NO:2. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%; 95%, 96%, 97%, 98%, or 99% identical to thepolynticleotide sequences encoding the polypeptides described above. Thepresent invention also encompasses the above polynucleotide sequencesfused to a heterologous polynucleotide sequence. Polypeptides encoded bythese polynucleotides are also encompassed by the invention.

[0144] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of an DR3-V1polypeptide, which may be described generally as having residues n1-m1of SEQ ID NO:2, where n1 and m1 are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0145] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of an extracellular domain of a protein results inmodification of loss of one or more biological functions of the protein,other functional activities (e.g., biological activities, ability tomultimerize, ability to bind DR3-V1 ligand) may still be retained. Forexample, the ability of shortened DR3-V1 extracellular domain muteins toinduce and/or bind to antibodies which recognize the complete, mature orextracellular domain forms of the polypeptides generally will beretained when less than the majority of the residues of the complete,mature or extracellular domain polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof an extracellular domain of a polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a DR3-V1extracellular domain mutein with a large number of deleted N-terminalamino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six DR3-V1extracellular domain amino acid residues may often evoke an immuneresponse.

[0146] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of theDR3-V1 extracellular domain amino acid sequence shown in SEQ ID NO:2, upto the cysteine residue at position number 206 and polynucleotidesencoding such polypeptides. In particular, the present inventionprovides polypeptides comprising, or alternatively consisting of, theamino acid sequence of residues n2-212 of SEQ ID NO:2, where n2 is aninteger from 36 to 206 corresponding to the position of the amino acidresidue in SEQ ID NO:2. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

[0147] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues Q-36 to F-212; G-37 to F-212; G-38 to F-212; T-39 toF-212; R-40 to F-212; S-41 to F-212; P-42 to F-212; R-43 to F-212; C-44to F-212; D-45 to F-212; C-46 to F-212; A-47 to F-212; G-48 to F-212;D-49 to F-212; F-50 to F-212; H-51 to F-212; K-52 to F-212; K-53 toF-212; I-54 to F-212; G-55 to F-212; L-56 to F-212; F-57 to F-212; C-58to F-212; C-59 to F-212; R-60 to F-212; G-61 to F-212; C-62 to F-212;P-63 to F-212; A-64 to F-212; G-65 to F-212; H-66 to F-212; Y-67 toF-212; L-68 to F-212; K-69 to F-212; A-70 to F-212; P-71 to F-212; C-72to F-212; T-73 to F-212; E-74 to F-212; P-75 to F-212; C-76 to F-212;G-77 to F-212; N-78 to F-212; S-79 to F-212; T-80 to F-212; C-81 toF-212; L-82 to F-212; V-83 to F-212; C-84 to F-212; P-85 to F-212; Q-86to F-212; D-87 to F-212; T-88 to F-212; F-89 to F-212; L-90 to F-212;A-91 to F-212; W-92 to F-212; E-93 to F-212; N-94 to F-212; H-95 toF-212; H-96 to F-212; N-97 to F-212; S-98 to F-212; E-99 to F-212; C-100to F-212; A-101 to F-212; R-102 to F-212; C-103 to F-212; Q-104 toF-212; A-105 to F-212; C-106 to F-212; D-107 to F-212; E-108 to F-212;Q-109 to F-212; A-110 to F-212; S-111 to F-212; Q-112 to F-212; V-113 toF-212; A-114 to F-212; L-115 to F-212; E-116 to F-212; N-117 to F-212;C-1 18 to F-212; S-119 to F-212; A-120 to F-212; V-121 to F-212; A-122to F-212; D-123 to F-212; T-124 to F-212; R-125 to F-212; C-126 toF-212; G-127 to F-212; C-128 to F-212; K-129 to F-212; P-130 to F-212;G-131 to F-212; W-132 to F-212; F-133 to F-212; V-134 to F-212; E-135 toF-212; C-136,to F-212; Q-137 to F-212; V-138 to F-212; S-139 to F-212;Q-140 to F-212; C-141 to F-212; V-142 to F-212; S-143 to F-212; S-144 toF-212; S-145 to F-212; P-146 to F-212; F-147 to F-212; Y-148 to F-212;C-149 to F-212; Q-150 to F-212; P-151 to F-212; C-152 to F-212; L-153 toF-212; D-154 to F-212; C-115 to F-212; G-156 to F-212; A-157 to F-212;L-158 to F-212; H-159 to F-212; R-160 to F-212; H-161 to F-212; T-162 toF-212; R-163 to F-212; L-164 to F-212; L-165 to F-212; C-166 to F-212;S-167 to F-212; R-168 to F-212; R-169 to F-212; D-170 to F-212; T-171 toF-212; D-172 to F-212; C-173 to F-212; G-174 to F-212; T-175 to F-212;C-176 to F-212; L-177 to F-212; P-178 to F-212; G-179 to F-212; F-180 toF-212; Y-181 to F-212; E-182 to F-212; H-183 to F-212; G-184 to F-212;D-185 to F-212; G-186 to F-212; C-187 to F-212; V-188 to F-212; S-189 toF-212; C-190 to F-212; P-191 to F-212; T-192 to F-212; S-193 to F-212;T-194 to F-212; L-195 to F-212; G-196 to F-212; S-197 to F-212; C-198 toF-212; P-199 to F-212; E-200 to F-212; R-201 to F-212; C-202 to F-212;A-203 to F-212; A-204 to F-212; V-205 to F-212; and C-206 to F-212 ofthe DR3-V1 sequence shown in SEQ ID NO:2. The present invention is alsodirected to nucleic acid molecules comprising, or alternativelyconsisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequencesencoding the polypeptides described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

[0148] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of an extracellular domain of a proteinresults in modification of loss of one or more biological functions ofthe protein, other functional activities (e.g., biological activities,ability to multimerize, ability to bind DR3-V1 ligand) may still beretained. For example the ability of the shortened DR3-V1 extracellulardomain mutein to induce and/or bind to antibodies which recognize thecomplete, mature or extracellular domain forms of the polypeptidegenerally will be retained when less than the majority of the residuesof the complete, mature or extracellular domain of a polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of an extracellular domain of a polypeptide retainssuch immunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata DR3-V1 extracellular domain mutein with a large number of deletedC-terminal amino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six DR3-V1extracellular domain amino acid residues may often evoke an immuneresponse.

[0149] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the extracellular domain of the DR3-V1polypeptide shown in SEQ ID NO:2, up to the proline residue at positionnumber 42, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues 36-m2of SEQ ID NO:2, where m2 is an integer from 42 to 212 corresponding tothe position of the amino acid residue in SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0150] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues Q-36 to F-212; Q-36 to M-21 1; Q-36 to Q-210; Q-36to R-209; Q-36 to W-208; Q-36 to G-207; Q-36 to C-206; Q-36 to V-205;Q-36 to A-204; Q-36 to A-203; Q-36 to C-202; Q-36 to R-201; Q-36 toE-200; Q-36 to P-199; Q-36 to C-198; Q-36 to S-197; Q-36 to G-196; Q-36to L-195; Q-36 to T-194; Q-36 to S-193; Q-36 to T-192; Q-36 to P-191;Q-36 to C-190; Q-36 to S-189; Q-36 to V-188; Q-36 to C-187; Q-36 toG-186; Q-36 to D-185; Q-36 to G-184; Q-36 to H-183; Q-36 to E-182; Q-36to Y-181; Q-36 to F-180; Q-36 to G-179; Q-36 to P-178; Q-36 to L-177;Q-36 to C-176; Q-36 to T-175; Q-36 to G-174; Q-36 to C-173; Q-36 toD-172; Q-36 to T-171; Q-36 to D-170; Q-36 to R-169; Q-36 to R-168; Q-36to S-167; Q-36 to C-166; Q-36 to L-165; Q-36 to L-164; Q-36 to R-163;Q-36 to T-162; Q-36 to H-161; Q-36 to R-160; Q-36 to H-159; Q-36 toL-158; Q-36 to A-157; Q-36 to G-156; Q-36 to C-155; Q-36 to D-154; Q-36to L-153; Q-36 to C-152; Q-36 to P-151; Q-36 to Q-150; Q-36 to C-149;Q-36 to Y-148; Q-36 to F-147; Q-36 to P-146; Q-36 to S-145; Q-36 toS-144; Q-36 to S-143; Q-36 to V-142; Q-36 to C-141; Q-36 to Q-140; Q-36to S-139; Q-36 to V-138; Q-36 to Q-137; Q-36 to C-136; Q-36 to E-135;Q-36 to V-134; Q-36 to F-133; Q-36 to W-132; Q-36 to G-131; Q-36 toP-130; Q-36 to K-129; Q-36 to C-128; Q-36 to G-127; Q-36 to C-126; Q-36to R-125; Q-36 to T-124; Q-36 to D-123; Q-36 to A-122; Q-36 to V-121;Q-36 to A-120; Q-36 to S-119; Q-36 to C-118; Q-36 to N-1 17; Q-36 toE-116; Q-36 to L-115; Q-36 to A-1 14; Q-36 to V-1 13; Q-36 to Q-112;Q-36 to S-111; Q-36 to A-110; Q-36 to Q-109; Q-36 to E-108; Q-36 toD-107; Q-36 to C-106; Q-36 to A-105; Q-36 to Q-104; Q-36 to C-103; Q-36to R-102; Q-36 to A-101; Q-36 to C-100; Q-36 to E-99; Q-36 to S-98; Q-36to N-97; Q-36 to H-96; Q-36 to H-95; Q-36 to N-94; Q-36 to E-93; Q-36 toW-92; Q-36 to A-91; Q-36 to L-90; Q-36 to F-89; Q-36 to T-88; Q-36 toD-87; Q-36 to Q-86; Q-36 to P-85; Q-36 to C-84; Q-36 to V-83; Q-36 toL-82; Q-36 to C-81; Q-36 to T-80; Q-36 to S-79; Q-36 to N-78; Q-36 toG-77; Q-36 to C-76; Q-36 to P-75; Q-36 to E-74; Q-36 to T-73; Q-36 toC-72; Q-36 to P-71; Q-36 to A-70; Q-36 to K-69; Q-36 to L-68; Q-36 toY-67; Q-36 to H-66; Q-36 to G-65; Q-36 to A-64; Q-36 to P-63; Q-36 toC-62; Q-36 to G-61; Q-36 to R-60; Q-36 to C-59; Q-36 to C-58; Q-36 toF-57; Q-36 to L-56; Q-36 to G-55; Q-36 to 1-54; Q-36 to K-53; Q-36 toK-52; Q-36 to H-51; Q-36 to F-50; Q-36 to D-49; Q-36 to G-48; Q-36 toA-47; Q-36 to C-46; Q-36 to D-45; Q-36 to C-44; Q-36 to R-43; and Q-36to P-42 of the sequence of the DR3-V1 sequence shown in SEQ ID NO:2. Thepresent invention is also directed to nucleic acid molecules comprising,or alternatively consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to thepolynucleotide sequences encoding the polypeptides described above. Thepresent invention also encompasses the above polynucleotide sequencesfused to a heterologous polynucleotide sequence. Polypeptides encoded bythese polynucleotides are also encompassed by the invention.

[0151] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of an DR3-V1polypeptide, which may be described generally as having residues n2-m2of SEQ ID NO:2, where n2 and m2 are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0152] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindDR3 ligand) may still be retained. For example, the ability of shortenedDR3 muteins to induce and/or bind to antibodies which recognize thecomplete or mature forms of the polypeptides generally will be retainedwhen less than the majority of the residues of the complete or maturepolypeptide are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a DR3 mutein with a large number of deleted N-terminalamino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six DR3 amino acidresidues may often evoke an immune response.

[0153] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the DR3amino acid sequence shown in SEQ ID NO:4, up to the arginine residue atposition number 412 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of the amino acid sequence of residues n3-417of SEQ ID NO:4, where n3 is an integer from 2 to 412 corresponding tothe position of the amino acid residue in SEQ ID NO:4. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

[0154] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues E-2 to P-417; Q-3 to P-417; R-4 to P-417; P-5 toP-417; R-6 to P-417; G-7 to P-417; C-8 to P-417; A-9 to P-417; A-10 toP-417; V-11 to P-417; A-12 to P-417; A-13 to P-417; A-14 to P-417; L-15to P-417; L-16 to P 417; L-17 to P-417; V-18 to P-417; L-19 to P-417;L-20 to P-417; G-21 to P-417; A-22 to P-417; R-23 to P-417; A-24 toP-417; Q-25 to P-417; G-26 to P-417; G-27 to P-417; T-28 to P-417; R-29to P-417; S-30 to P-417; P-31 to P-417; R-32 to P-417; C-33 to P-417;D-34 to P-417; C-35 to P-417; A-36 to P-417; G-37 to P-417; D-38 toP-417; F-39 to P-417; H-40 to P-417; K-41 to P-417; K-42 to P-417; I-43to P-417; G-44 to P-417; L-45 to P-417; F-46 to P-417; C-47 to P-417;C-48 to P-417; R-49 to P-417; G-50 to P-417; C-51 to P-417; P-52 toP-417; A-53 to P-417; G-54 to P417; H-55 to P-417; Y-56 to P-417; L-57to P-417; K-58 to P-417; A-59 to P-417; P-60 to P-417; C-61 to P-417;T-62 to P-417; E-63 to P-417; P-64 to P-417; C-65 to P-417; G-66 toP-417; N-67 to P-417; S-68 to P-417; T-69 to P-417; C-70 to P-417; L-71to P-417; V-72 to P-417; C-73 to P-417; P-74 to P-417; Q-75 to P-417;D-76 to P-417; T-77 to P417; F-78 to P-417; L-79 to P-417; A-80 toP-417; W-81 to P-417; E-82 to P-417; N-83 to P-417; H-84 to P-417; H-85to P-417; N-86 to P-417; S-87 to P-417; E-88 to P-417; C-89 to P-417;A-90 to P-417; R-91 to P-417; C-92 to P-417; Q-93 to P-417; A-94 toP-417; C-95 to P-417; D-96 to P-417; E-97 to P-417; Q-98 to P-417; A-99to P-417; S-100 to P-417; Q-101 to P-417; V-102 to P-417; A-103 toP-417; L-104 to P-417; E-105 to P-417; N-106 to P-417; C- 107 to P-417;S-108 to P-417; A-109 to P-417; V-110 to P-417; A-111 to P-417; D -112to P-417; T-113 to P-417; R-114 to P-417; C-115 to P-417; G-116 toP-417; C-117 to P-417; K-118 to P-417; P-119 to P-417; G-120 to P-417;W-121 to P-417; F-122 to P-417; V-123 to P-417; E-124 to P-417; C-125 toP-417; Q-126 to P-417; V-127 to P-417; S-128 to P-417; Q-129 to P-417;C-130 to P-417; V-131 to P-417; S-132 to P-417; S-133 to P-417; S-134 toP-417; P-135 to P-417; F-136 to P-417; Y-137 to P-417; C-138 to P-417;Q-139 to P-417; P-140 to P-417; C-141 to P-417; L-142 to P-417; D-143 toP-417; C-144 to P-417; G-145 to P-417; A-146 to P-417; L-147 to P-417;H-148 to P-417; R-149 to P-417; H-150 to P-417; T-151 to P-417; R-152 toP-417; L-153 to P-417; L-154 to P-417; C-155 to P-417; S-156 to P-417;R-157 to P-417; R-158 to P-417; D-159 to P-417; T-160 to P-417; D-161 toP-417;.C-162 to P-417; G-163 to P-417; T-164 to P-417; C-165 to P-417;L-166 to P-417; P-167 to P-417; G-168 to P-417; F-169 to P-417; Y-170 toP-417; E-171 to P-417; H-172 to P-417; G-173 to P-417; D-174 to P-417;G-175 to P-417; C-176 to P-417; V-177 to P-417; S-178 to P-417; C-179 toP-417; P-180 to P-417; T-181 to P-417; S-182 to P-417; T-183 to P-417;L-184 to P-417; G-185 to P-417; S-186 to P-417; C-187 to P-417; P-188 toP-417; E-189 to P-417; R-190 to P-417; C-191 to P-417; A-192 to P-417;A-193 to P-417; V-194 to P-417; C-195 to P-417; G-196 to P417; W-197 toP-417; R-198 to P-417; Q-199 to P-417; M-200 to P-417; F-201 to P-417;W-202 to P-417; V-203 to P-417; Q-204 to P-417; V-205 to P-417; L-206 toP-417; L-207 to P-417; A-208 to P-417; G-209 to P-417; L-210 to P-417;V-211 to P-417; V-212 to P-417; P-213 to P-417; L-214 to P-417; L-215 toP-417; L-216 to P-417; G-217 to P-417; A-218 to P-417; T-219 to P-417;L-220 to P-417; T-221 to P-417; Y-222 to P-417; T-223 to P-417; Y-224 toP-417; R-225 to P-417; H-226 to P-417; C-227 to P-417; W-228 to P-417;P-229 to P-417; H-230 to P-417; K-231 to P-417; P-232 to P-417; L-233 toP-417; V-234 to P-417; T-235 to P-417; A-236 to P-417; D-237 to P-417;E-238 to P-417; A-239 to P-417; G-240 to P-417; M-241 to P-417; E-242 toP-417; A-243 to P-417; L-244 to P-417; T-245 to P-417; P-246 to P-417;P-247 to P-417; P-248 to P-417; A-249 to P-417; T-250 to P-417; H-251 toP-417; L-252 to P-417; S-253 to P-417; P-254 to P-417; L-255 to P-417;D-256 to P-417; S-257 to P-417; A-258 to P-417; H-259 to P-417; T-260 toP-417; L-261 to P-417; L-262 to P-417; A-263 to P-417; P-264 to P-417;P-265 to P-417; D-266 to P-417; S-267 to P-417; S-268 to P-417; E-269 toP-417; K-270 to P-417; 1-271 to P-417; C-272 to P-417; T-273 to P-417;V-274 to P-417; Q-275 to P-417; L-276 to P-417; V-277 to P-417; G-278 toP-417; N-279 to P-417; S-280 to P-417; W-281 to P-417; T-282 to P-417;P-283 to P-417; G-284 to P-417; Y-285 to P-417; P-286 to P-417; E-287 toP-417; T-288 to P-417; Q-289 to P-417; E-290 to P-417; A-291 to P-417;L-292 to P-417; C-293 to P-417; P-294 to P-417; Q-295 to P-417; V-296 toP-417; T-297 to P-417; W-298 to P-417; S-299 to P-417; W-300 to P-417;D-301 to P-417; Q-302 to P-417; L-303 to P-417; P-304 to P-417; S-305 toP-417; R-306 to P-417; A-307 to P-417; L-308 to P-417; G-309 to P-417;P-310 to P-417; A-311 to P-417; A-312 to P-417; A-313 to P-417; P-314 toP-417; T-315 to P-417; L-316 to P-417; S-317 to P417; P-318 to P417;E-319 to P-417; S-320 to P-417; P-321 to P-413; A-322 to P417; G-323 toP-417; S-324 to P-417; P-325 to P-417; A-326 to P-417; M-327 to P-417;M-328 to P-417; L-329 to P-417; Q-330 to P-417; P-331 to P-417; G-332 toP-417; P-333 to P-417; Q-334 to P-417; L-335 to P-417; Y-336 to P-417;D-337 to P-417; V-338 to P-417; M-339 to P-417; D-340 to P-417; A-341 toP-417; V-342 to P-417; P-343 to P-417; A-344 to P-417; R-345 to P-417;R-346 to P-417; W-347 to P-417; K-348 to P-417; E-349 to P-417; F-350 toP-417; V-351 to P-417; R-352 to P-417; T-353 to P-417; L-354 to P-417;G-355 to P-417; L-356 to P-417; R-357 to P-417; E-358 to P-417; A-359 toP-417; E-360 to P417; 1-361 to P-417; E-362 to P-417; A-363 to P-417;V-364 to P-417; E-365 to P-417; V-366 to P-417; E-367 to P417; 1-368 toP-417; G-369 to P-417; R-370 to P-417; F-371 to P-417; R-372 to P-417;D-373 to P-417; Q-374 to P-417; Q-375 to P-417; Y-376 to P-417; E-377 toP-417; M-378 to P-417; L-379 to P-417; K-380 to P-417; R-381 to P-417;W-382 to P-417; R-383 to P-417; Q-384 to P-417; Q-385 to P-417; Q-386 toP-417; P-387 to P-417; A-388 to P-417; G-389 to P-417; L-390 to P-417;G-391 to P-417; A-392 to P-417; V-393 to P-417; Y-394 to P-417; A-395 toP-417; A-396 to P-417; L-397 to P-417; E-398 to P-417; R-399 to P-417;M-400 to P-417; G-401 to P-417; L-402 to P-417; D-403 to P417; G-404 toP-417; C-405 to P-417; V-406 to P-417; E-407 to P-417; D-408 to P-417;L-409 to P-417; R-410 to P-417; S-411 to P-417; and R-412 to P-417 ofthe DR3 sequence shown in SEQ ID NO:4. The present invention is alsodirected to nucleic acid molecules comprising, or alternativelyconsisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequencesencoding the polypeptides described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

[0155] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of a protein results in modification of lossof one or more biological functions of the protein, other functionalactivities (e.g., biological activities, ability to multimerize, abilityto bind DR3 ligand) may still be retained. For example the ability ofthe shortened DR3 mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a DR3 mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six DR3amino acid residues may often evoke an immune response.

[0156] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the DR3 polypeptide shown in SEQ ID NO:4, up tothe arginine residue at position number 6, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues 1-m3 of SEQ ID NO:4, where m3 is an integer from 6to 416 corresponding to the position of the amino acid residue in SEQ IDNO:4. Polynucleotides encoding these polypeptides are also encompassedby the invention.

[0157] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues M-1 to G-416; M-1 to R-415; M-1 to Q-414; M-1 toL-413; M-1 to R-412; M-1 to S-411; M-1 to R-410; M-1 to L-409; M-1 toD-408; M-1 to E-407; M-1 to V-406; M-1 to C-405; M-1 to G-404; M-1 toD-403; M-1 to L-402; M-1 to G-401; M-1 to M-400; M-1 to R-399; M-1 toE-398; M-1 to L-397; M-1 to A-396; M-1 to A-395; M-1 to Y-394; M-1 toV-393; M-1 to A-392; M-1 to G-391; M-1 to L-390; M-1 to G-389; M-1 toA-388; M-1 to P-387; M-1 to Q-386; M-1 to Q-385; M-1 to Q-384; M-1 toR-383; M-1 to W-382; M-1 to R-381; M-1 to K-380; M-1 to L-379; M-1 toM-378; M-1 to E-377; M-1 to Y-376; M-1 to Q-375; M-1 to Q-374; M-1 toD-373; M-1 to R-372; M-1 to F-371; M-1 to R-370; M-1 to G-369; M-1 toI-368; M-1 to E-367; M-1 to V-366; M-1 to E-365; M-1 to V-364; M-1 toA-363; M-1 to E-362; M-1 to 1-361; M-1 to E-360; M-1 to A-359; M- 1 toE-358; M-1 to R-357; M- 1 to L-356; M- 1 to G-355; M-1 to L-354; M-1 toT-353; M-1 to R-352; M-1 to V-351; M-1 to F-350; M-1 to E-349; M-1 toK-348; M-1 to W-347; M-1 to R-346; M-1 to R-345; M-1 to A-344; M-1 toP-343; M-1 to V-342; M-1 to A-341; M-1 to D-340; M-1 to M-339; M-1 toV-338; M-1 to D-337; M-1 to Y-336; M-1 to L-335; M-1 to Q-334; M-1 toP-333; M-1 to G-332; M-1 to P-331; M-1 to Q-330; M-1 to L-329; M-1 toM-328; M-1 to M-327; M-1 to A-326; M-1 to P-325; M-1 to S-324; M-1 toG-323; M-1 to A-322; M-1 to P-321; M-1 to S-320; M-1 to E-319; M-1 toP-318; M-1 to S-317; M-1 to L-316; M-1 to T-315; M-1 to P-314; M-1 toA-313; M-1 to A-312; M-1 to A-311; M-1 to P-310; M-1 to G-309; M-1 toL-308; M-1 to A-307; M-1 to R-306; M-1 to S-305; M-1 to P-304; M-1 toL-303; M-1 to Q-302; M-1 to D-301; M-1 to W-300; M-1 to S-299; M-1 toW-298; M-1 to T-297; M-1 to V-296; M-1 to Q-295; M-1 to P-294; M-1 toC-293; M-1 to L-292; M-1 to A-291; M-1 to E-290; M -1 to Q-289; M -1 toT-288; M -1 to E-287; M-1 to P-286; M-1 to Y-285; M-1 to G-284; M-1 toP-283; M-1 to T-282; M-1 to W-281; M-1 to S-280; M-1 to N-279; M-1 toG-278; M-1 to V-277; M-1 to L-276; M-1 to Q-275; M-1 to V-274; M-1 toT-273; M-1 to C-272; M-1 to I-271; M-1 to K-270; M-1 to E-269; M-1 toS-268; M-1 to S-267; M-1 to D-266; M-1 to P-265; M-1 to P-264; M-1 toA-263; M-1 to L-262; M-1 to L-261; M-1 to T-260; M-1 to H-259; M-1 toA-258; M-1 to S-257; M-1 to D-256; M-1 to L-255; M-1 to P-254; M-1 toS-253; M-1 to L-252; M-1 to H-25 1; M-1 to T-250; M-1 to A-249; M-1 toP-248; M-1 to P-247; M-1 to P-246; M-1 to T-245; M-1 to L-244; M-1 toA-243; M-1 to E-242; M-1 to M-241; M-1 to G-240; M-1 to A-239; M-1 toE-238; M-1 to D-237; M-1 to A-236; M-1 to T-235; M-1 to V-234; M-1 toL-233; M-1 to P-232; M-1 to K-231; M-1 to H-230; M-1 to P-229; M-1 toW-228; M-1 to C-227; M-1 to H-226; M-1 to R-225; M-1 to Y-224; M-1 toT-223; M-1 to Y-222; M-1 to T-221; M-1 to L-220; M-1 to T-219; M-1 toA-218; M-1 to G-217; M-1 to L-216; M-1 to L-215; M-1 to L-214; M-1 toP-213; M-1 to V-212; M-1 to V-211; M-1 to L-210; M-1 to G-209; M-1 toA-208; M-1 to L-207; M-1 to L-206; M-1 to V-205; M-1 to Q-204; M-1 toV-203; M -1 to W-202; M-1 to F-201; M-1 to M-200; M-1 to Q-199; M-1 toR-198; M-1 to W-197; M-1 to G-196; M-1 to C-195; M-1 to V-194; M-1 toA-193; M-1 to A-192; M-1 to C-191; M-1 to R-190; M-1 to E-189; M-1 toP-188; M-1 to C-187; M-1 to S-186; M-1 to G-185; M-1 to L-184; M-1 toT-183; M-1 to S-182; M-1 to T-181; M-1 to P-180; M-1 to C-179; M-1 toS-178; M-1 to V-177; M-1 to C-176; M-1 to G-175; M-1 to D-174; M-1 toG-173; M-1 to H-172; M-1 to E-171; M-1 to Y-170; M-1 to F-169; M-1 toG-168; M-1 to P-167; M-1 to L-166; M-1 to C-165; M-1 to T-164; M-1 toG-163; M-1 to C-162; M-1 to D-161; M-1 to T-160; M-1 to D-159; M-1 toR-158; M-1 to R-157; M-1 to S-156; M-1 to C-155; M-1 to L-154; M-1 toL-153, M-1 to R-152; M-1 to T-151; M-1 to H-150; M-1 to R-149; M-1 toH-148; M-1 to L-147; M-1 to A-146; M-1 to G-145; M-1 to C-144; M-1 toD-143; M-1 to L-142; M-1 to C-141; M-1 to P-140; M-1 to Q-139;M-1 toC-138; M-1 to Y-137; M-1 to F-136; M-1 to P-135; M-1 to S-134; M-1 toS-133; M-1 to S-132; M-1 to V-131; M-1 to C-130; M-1 to Q-129; M-1 toS-128; M-1 to V-127; M-1 to Q-126; M-1 to C-125; M-1 to E-124; M-1 toV-123; M-1 to F-122; M-1 to W-121; M-1 to G-120; M-1 to P-119; M-1 toK-118; M-1 to C-117; M-1 to G-116; M-1 to C-115; M-1 to R-114; M-1 toT-113; M-1 to D-112; M-1 to A-111; M-1 to V-110; M-1 to A-109; M-1 toS-108; M-1 to C-107; M-1 to N-106; M-1 to E-105, M-1 to L-104; M-1 toA-103; M-1 to V-102; M-1 to Q-101; M-1 to S-100; M-1 to A-99; M-1 toQ-98; M-1 to E-97; M-1 to D-96; M-1 to C-95; M-1 to A-94; M-1 to Q-93;M-1 to C-92; M-1 to R-91; M-1 to A-90; M-1 to C-89; M-1 to E-88; M-1 toS-87; M-1 to N-86; M-1 to H-85; M-1 to H-84; M-1 to N-83; M-1 to E-82;M-1 to W-81; M-1 to A-80; M-1 to L-79; M-1 to F-78; M-1 to T-77; M-1 toD-76; M-1 to Q-75; M-1 to P-74; M-1 to C-73; M-1 to V-72; M-1 to L-71;M-1 to C-70; M-1 to T-69; M-1 to S-68; M-1 to N-67; M-1 to G-66; M-1 toC-65; M-1 to P-64; M-1 to E-63; M-1 to T-62; M-1 to C-61; M-1 to P-60;M-1 to A-59; M-1 to K-58; M-1 to L-57; M-1 to Y-56; M-1 to H-55; M-1 toG-54; M-1 to A-53; M-1 to P-52; M-1 to C-51; M-1 to G-50; M-1 to R-49;M-1 to C-48; M-1 to C-47; M-1 to F-46; M-1 to L-45; M-1 to G-44; M-1 to1-43; M-1 to K-42; M-1 to K-41; M-1 to H-40; M-1 to F-39; M-1 to D-38;M-1 to G-37; M-1 to A-36; M-1 to C-35; M-1 to D-34; M-1 to C-33; M-1 toR-32; M-1 to P-31; M-1 to S-30; M-1 to R-29; M-1 to T-28; M-1 to G-27;M-1 to G-26; M-1 to Q-25; M-1 to A-24; M-1 to R-23; M-1 to A-22; M-1 toG-21; M-1 to L-20; M-1 to L-19; M-1 to V-18; M-1 to L-17; M-1 to L-16;M-1 to L-15; M-1 to A-14; M-1 to A-13; M-1 to A-12; M-1 to V-11; M-1 toA-10; M-1 to A-9; M-1 to C-8; M-1 to G-7; and M-1 to R-6 of the sequenceof the DR3 sequence shown in SEQ ID NO:4. The present invention is alsodirected to nucleic acid molecules comprising, or alternativelyconsisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequencesencoding the polypeptides described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

[0158] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of an DR3polypeptide, which may be described generally as having residues n3-m3of SEQ ID NO:4, where n3 and m3 are integers as described above.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0159] As mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of an extracellular domain of a protein results inmodification of loss of one or more biological functions of the protein,other functional activities (e.g., biological activities, ability tomultimerize, ability to bind DR3 ligand) may still be retained. Forexample, the ability of shortened DR3 extracellular domain muteins toinduce and/or bind to antibodies which recognize the complete, mature orextracellular domain forms of the polypeptides generally will beretained when less than the majority of the residues of the complete,mature or extracellular domain polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a DR3 extracellular domainmutein with a large number of deleted N-terminal amino acid residues mayretain some biological or immunogenic activities. In fact, peptidescomposed of as few as six DR3 extracellular domain amino acid residuesmay often evoke an immune response.

[0160] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the amino terminus of the DR3extracellular domain amino acid sequence shown in SEQ ID NO:4, up to thecysteine residue at position number 195 and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising, or alternatively consisting of, the amino acidsequence of residues n4-201 of SEQ ID NO:4, where n4 is an integer from25 to 195 corresponding to the position of the amino acid residue in SEQID NO:4. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0161] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues Q-25 to F-201; G-26 to F-201; 6-27 to F-201; T-28 toF-201; R-29 to F-201; S-30 to F-201; P-31 to F-201; R-32 to F-201; C-33to F-201; D-34 to F-201; C-35 to F-201; A-36 to F-201; G-37 to F-201;D-38 to F-201; F-39 to F-201; H-40 to F-201; K-41 to F-201; K-42 toF-201; 1-43 to F-201; G-44 to F-201; L-45 to F-201; F-46 to F-201; C-47to F-201; C-48 to F-201; R-49 to F-201; G-50 to F-201; C-51 to F-201;P-52 to F-201; A-53 to F-201; G-54 to F-201; H-55 to F-201; Y-56 toF-201; L-57 to F-201; K-58 to F-201; A-59 to F-201; P-60 to F-201; C-61to F-201; T-62 to F-201; E-63 to F-201; P-64 to F-201; C-65 to F-201;G-66 to F-201; N-67 to F-201; S-68 to F-201; T-69 to F-201; C-70 toF-201; L-71 to F-201; V-72 to F-201; C-73 to F-201; P-74 to F-201; Q-75to F-201; D-76 to F-201; T-77 to F-201; F-78 to F-201; L-79 to F-201;A-80 to F-201; W-81 to F-201; E-82 to F-201; N-83 to F-201; H-84 toF-201; H-85 to F-201; N-86 to F-201; S-87 to F-201; E-88 to F-201; C-89to F-201; A-90 to F-201; R-91 to F-201; C-92 to F-201; Q-93 to F-201;A-94 to F-201; C-95 to F-201; D-96 to F-201; E-97 to F-201; Q-98 toF-201; A-99 to F-201; S-100 to F-201; Q-101 to F-201; V-102 to F-201;A-103 to F-201; L-104 to F-201; E-105 to F-201; N-106 to F-201; C-107 toF-201; S-108 to F-201; A-109 to F-201; V-110 to F-201; A-111 to F-201;D-112 to F-201; T-113 to F-201; R-114 to F-201; C-115 to F-201; G-116 toF-201; C-117 to F-201; K-118 to F-201; P-119 to F-201; G-120 to F-201;W-121 to F-201; F-122 to F-201; V-123 to F-201; E-124 to F-201; C-125 toF-201; Q-126 to F-201; V-127 to F-201; S-128 to F-201; Q-129 to F-201;C-130 to F-201; V-131 to F-201; S-132 to F-201; S-133 to F-201; S-134 toF-201; P-135 to F-201; F-136 to F-201; Y-137 to F-201; C-138 to F-201;Q-139 to F-201; P-140 to F-201; C-141 to F-201; L-142 to F-201; D-143 toF-201; C-144 to F-201; G-145 to F-201; A-146 to F-201; L-147 to F-201;H-148 to F-201; R-149 to F-201; H-150 to F-201; T-151 to F-201; R-152 toF-201; L-153 to F-201; L-154 to F-201; C-155 to F-201; S-156 to F-201;R-157 to F-201; R-158 to F-201; D-159 to F-201; T-160 to F-201; D-161 toF-201; C-162 to F-201; G-163 to F-201; T-164 to F-201; C-165 to F-201;L-166 to F-201; P-167 to F-201; G-168 to F-201; F-169 to F-201; Y-170 toF-201; E-171 to F-201; H-172 to F-201; G-173 to F-201; D-174 to F-201;G-175 to F-201; C-176 to F-201; V-177 to F-201; S-178 to F-201; C-179 toF-201; P-180 to F-201; T-181 to F-201; S-182 to F-201; T-183 toF-201;L-184 to F-201; G-185 to F-201; S-186 to F-201; C-187 to F-201;P-188 to F-201; E-189 to F-201; R-190 to F-201; C-191 to F-201; A-192 toF-201; A-193 to F-201; V-194 to F-201; and C-195 to F-201 of the DR3sequence shown in SEQ ID NO:4. The present invention is also directed tonucleic acid molecules comprising, or alternatively consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,or 99% identical to the polynucleotide sequences encoding thepolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

[0162] Also as mentioned above, even if deletion of one or more aminoacids from the C-terminus of an extracellular domain of a proteinresults in modification of loss of one or more biological functions ofthe protein, other functional activities (e.g., biological activities,ability to multimerize, ability to bind DR3 ligand) may still beretained. For example the ability of the shortened DR3 extracellulardomain mutein to induce and/or bind to antibodies which recognize thecomplete or mature forms of the polypeptide generally will be retainedwhen less than the majority of the residues of the complete or maturepolypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a DR3 extracellular domain mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six DR3extracellular domain amino acid residues may often evoke an immuneresponse.

[0163] Accordingly, the present invention further provides polypeptideshaving one or more residues deleted from the carboxy terminus of theamino acid sequence of the DR3 extracellular domain polypeptide shown inSEQ ID NO:4, up to the proline residue at position number 31, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising, or alternatively consistingof, the amino acid sequence of residues 1-m4 of SEQ ID NO:4, where m4 isan integer from 31 to 201 corresponding to the position of the aminoacid residue in SEQ ID NO:4. Polynucleotides encoding these polypeptidesare also encompassed by the invention.

[0164] More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of a member selected from the group consisting ofamino acid residues Q-25 to F-201; Q-25 to M-200; Q-25 to Q-199; Q-25 toR-198; Q-25 to W-197; Q-25 to G-196; Q-25 to C-195; Q-25 to V-194; Q-25to A-1 93; Q-25 to A-192; Q-25 to C-191; Q-25 to R-190; Q-25 to E-189;Q-25 to P-188; Q-25 to C-187; Q-25 to S-186; Q-25 to G-185; Q-25 toL-184; Q-25 to T-183; Q-25 to S-182; Q-25 to T-181; Q-25 to P-180; Q-25to C-179; Q-25 to S-178; Q-25 to V-177; Q-25 to C-176; Q-25 to G-175;Q-25 to D-174; Q-25 to G-173; Q-25 to H-1 72; Q-25 to E-171; Q-25 toY-170; Q-25 to F-169; Q-25 to G-168; Q-25 to P-167; Q-25 to L-166; Q-25to C-165; Q-25 to T-164; Q-25 to G-163; Q-25 to C-162; Q-25 to D-161;Q-25 to T-160; Q-25 to D-159; Q-25 to R-158; Q-25 to R-157; Q-25 toS-156; Q-25 to C-155; Q-25 to L-154; Q-25 to L-153; Q-25 to R-152; Q-25to T-151; Q-25 to H-150; Q-25 to R-149; Q-25 to H-148; Q-25 to L-147;Q-25 to A-146; Q-25 to G-145; Q-25 to C-144; Q-25 to D-143; Q-25 toL-142; Q-25 to C-141; Q-25 to P-140; Q-25 to Q-139; Q-25 to C-138; Q-25to Y-137; Q-25 to F-136; Q-25 to P-135; Q-25 to S-134; Q-25 to S-133;Q-25 to S-132; Q-25 to V-131; Q-25 to C-130; Q-25 to Q-129; Q-25 toS-128; Q-25 to V-127; Q-25 to Q-126; Q-25 to C-125; Q-25 to E-124; Q-25to V-123; Q-25 to F-122; Q-25 to W-121; Q-25 to G-120; Q-25 to P-119;-Q-25 to K-118; Q-25 to C-117; Q-25 to G-116; Q-25 to C-115; Q-25 toR-114; Q-25 to T-113; Q-25 to D-112; Q-25 to A-111; Q-25 to V-110; Q-25to A-109; Q-25 to S-108; Q-25 to C-107; Q-25 to N-106; Q-25 to E-105;Q-25 to L-104; Q-25 to A-103; Q-25 to V-102; Q-25 to Q-101; Q-25 toS-100; Q-25 to A-99; Q-25 to Q-98; Q-25 to E-97; Q-25 to D-96; Q-25 toC-95; Q-25 to A-94; Q-25 to Q-93; Q-25 to C-92; Q-25 to R-91; Q-25 toA-90; Q-25 to C-89; Q-25 to E-88; Q-25 to S-87; Q-25 to N-86; Q-25 toH-85; Q-25 to H-84; Q-25 to N-83; Q-25 to E-82; Q-25 to W-81; Q-25 toA-80; Q-25 to L-79; Q-25 to F-78; Q-25 to T-77; Q-25 to D-76; Q-25 toQ-75; Q-25 to P-74; Q-25 to C-73; Q-25 to V-72; Q-25 to L-71; Q-25 toC-70; Q-25 to T-69; Q-25 to S-68; Q-25 to N-67; Q-25 to G-66; Q-25 toC-65; Q-25 to P-64; Q-25 to E-63; Q-25 to T-62; Q-25 to C-61; Q-25 toP-60; Q-25 to A-59; Q-25 to K-58; Q-25 to L-57; Q-25 to Y-56; Q-25 toH-55; Q-25 to G-54; Q-25 to A-53; Q-25 to P-52; Q-25 to C-51; Q-25 toG-50; Q-25 to R-49; Q-25 to C-48; Q-25 to C-47; Q-25 to F-46; Q-25 toL-45; Q-25 to G-44; Q-25 to 1-43; Q-25 to K42; Q-25 to K-41; Q-25 toH-40; Q-25 to F-39; Q-25 to D-38; Q-25 to G-37; Q-25 to A-36; Q-25 toC-35; Q-25 to D-34; Q-25 to C-33; Q-25 to R-32; and Q-25 to P-31 of thesequence of the DR3 sequence shown in SEQ ID NO:4. The present inventionis also directed to nucleic acid molecules comprising, or alternativelyconsisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequencesencoding the polypeptides described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

[0165] The invention also provides polypeptides having one or more aminoacids deleted from both the amino and the carboxyl termini of a DR3extracellular domain polypeptide, which may be described generally ashaving residues n4-m4 of SEQ ID NO:4, where n4 and m4 are integers asdescribed above. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0166] The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the DR3 polypeptide sequence set forth herein as n1-m1,n2-m2, n3-m3, and/or n4-m4. In preferred embodiments, the application isdirected to proteins containing polypeptides at least 80%, 85%, 90%,92%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having theamino acid sequence of the specific DR3 N- and C-terminal deletionsrecited herein. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0167] In certain preferred embodiments, DR3 proteins of the inventioncomprise, or alternatively consist of, fusion proteins as describedabove wherein the DR3 polypeptides are those described as n1-m1, n2-m2,n3-m3, and/or n4-m4 herein. In preferred embodiments, the application isdirected to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98% or 99% identical to the nucleic acid sequences encodingpolypeptides having the amino acid sequence of the specific N- andC-terminal deletions recited herein. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

[0168] It is believed one or more of the cysteine rich regions of DR3-V1and DR3 are important for interactions between DR3-V1 and DR3 and theirrespective ligands. Accordingly, specific embodiments of the inventionare directed to polynucleotides encoding polypeptides which comprise, oralternatively consist of, the amino acid sequence of amino acid residues58 to 103, 106 to 136, 141 to 173, or 176 to 206 of SEQ ID NO:2.Additional embodiments of the invention are directed to polynucleotidesencoding DR3-V1 or DR3 polypeptides which comprise, or alternativelyconsist of, any combination of 1, 2, 3, or all 4 of the cysteine richregions described above. Polypeptides encoded by these polynucleotidesare also encompassed by the invention.

[0169] Preferably, the polynucleotide fragments of the invention encodea polypeptide which demonstrates a DR3 functional activity. By apolypeptide demonstrating a DR3-V1 or DR3 “functional activity” ismeant, a polypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) DR3-V1 or DR3protein. Such functional activities include, but are not limited to,biological activity (e.g., ability to induce apoptosis), antigenicity(the ability to bind, or compete for binding with a DR3-V1 or DR3polypeptide for binding, to an anti-DR3-V1 or anti-DR3 antibody),immunogenicity (ability to generate antibody which binds to a DR3-V1 orDR3 polypeptide), ability to form multimers with DR3-V1 or DR3polypeptides of the invention, and ability to bind to a receptor orligand for a DR3-V1 or DR3 polypeptide (e.g., TNF-γ, TNF-γ-β).

[0170] The functional activity of DR3-V1 or DR3 polypeptides, andfragments, variants derivatives, and analogs thereof, can be assayed byvarious methods.

[0171] For example, in one embodiment where one is assaying for theability to bind or compete with full-length DR3-V1 or DR3 polypeptidefor binding to anti-DR3-V1 or anti-DR3 antibody, various immunoassaysknown in the art can be used, including but not limited to, competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions; agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

[0172] In another embodiment, where a DR3-V1 or DR3 ligand isidentified, or the ability of a polypeptide fragment, variant orderivative of the invention to multimerize is being evaluated, bindingcan be assayed, e.g., by means well-known in the art, such as, forexample, reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., Microbiol. Rev. 59:94-123 (1995). In another embodiment,physiological correlates of DR3-V1 or DR3 binding to its substrates(signal transduction) can be assayed.

[0173] In addition, assays described herein (see Example 6) andotherwise known in the art may routinely be applied to measure theability of DR3-V1 or DR3 polypeptides and fragments, variantsderivatives and analogs thereof to elicit DR3-V1 or DR3 relatedbiological activity (e.g., to induce apoptosis in vitro or in vivo). Theability of polynucleotides and polypeptides of the invention to increaseor decrease apoptosis can routinely be determined using techniques knownin the art. For example, biological activity can routinely be measuredusing cell death assays performed essentially as previously described(Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol.Chem. 270:7795-8(1995); Kischkel et al., EMBO 14:5579-5588 (1995);Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)).

[0174] It is believed one or more of the cysteine rich regions of DR3-V1or DR3 is important for interactions between DR3-V1 or DR3 and itsligands. Accordingly, specific embodiments of the invention are directedto polypeptides which comprise, or alternatively consist of the aminoacid sequence of amino acid residues 58 to 103, 106 to 136, 141 to 173,or 176 to 206 of SEQ ID NO:2. Additional embodiments of the inventionare directed to polypeptides which comprise, or alternatively consistof, any combination of 1, 2, 3, or all 4 of the cysteine rich regionsdescribed above. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

[0175] Among the especially preferred fragments of the invention arefragments characterized by structural or functional attributes of DR3-V1or DR3. Such fragments include amino acid residues that comprisealpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet-forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, surface forming regions,and high antigenic index regions (i.e., containing four or morecontiguous amino acids having an antigenic index of greater than orequal to 1.5, as identified using the default parameters of theJameson-Wolf program) of complete (i.e., full-length) DR3-V 1 or DR3(SEQ ID NO:2 or SEQ ID NO:4). Certain preferred regions are those setout in FIG. 4 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in SEQ ID NO:2 or SEQ ID NO:4, such preferred regions include;Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Chou-Fasman predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic andhydrophobic regions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

[0176] In additional embodiments, the polynucleotides of the inventionencode functional attributes of DR3-V1 or DR3. Preferred embodiments ofthe invention in this regard include fragments that comprise alpha-helixand alpha-helix forming regions (“alpha-regions”, beta-sheet andbeta-sheet forming regions (“beta-regions”), turn and turn-formingregions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of DR3-V1 orDR3.

[0177] The data representing the structural or functional attributes ofDR3-V1 or DR3 set forth in FIG. 4 and/or Table 2, as described above,was generated using the various modules and algorithms of the DNA*STARset on default parameters. In a preferred embodiment, the data presentedin columns VIII, IX, XIII, and XIV of Table 2 can be used to determineregions of DR3-V1 which exhibit a high degree of potential forantigenicity. Regions of high antigenicity are determined from the datapresented in columns VIII, IX, XIII, and/or IV by choosing values whichrepresent regions of the polypeptide which are likely to be exposed onthe surface of the polypeptide in an environment in which antigenrecognition may occur in the process of initiation of an immuneresponse.

[0178] Certain preferred regions in these regards are set out in FIG. 4,but may, as shown in Table 2, be represented or identified by usingtabular representations of the data presented in FIG. 4. The DNA*STARcomputer algorithm used to generate FIG. 4 (set on the original defaultparameters) was used to present the data in FIG. 4 in a tabular format(See Table 2). The tabular format of the data in FIG. 4 may be used toeasily determine specific boundaries of a preferred region.

[0179] The above-mentioned preferred regions set out in FIG. 4 and inTable 2 include, but are not limited to, regions of the aforementionedtypes identified by analysis of the amino acid sequence set out in SEQID NO:2. As set out in FIG. 4 and in Table 2, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions (columns I, III, V, and VII in Table 2), Chou-Fasmanalpha-regions, beta-regions, and turn-regions (columns II, IV, and VI inTable 2), Kyte-Doolittle hydrophilic regions (column VIII in Table 2),Hopp-Woods hydrophobic regions (column IX in Table 2), Eisenberg alpha-and beta-amphipathic regions (columns X and XI in Table 2),Karplus-Schulz flexible regions (column XII in Table 2), Jameson-Wolfregions of high antigenic index (column XIII in Table 2), and Eminisurface-forming regions (column XIV in Table 2). TABLE 2 Res Position III III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . . . . . . 1.03−0.61 . . . 0.95 1.60 Glu 2 A . . . . . . 1.42 −0.64 . . . 0.95 2.17 Glu3 A . . . . . . 1.47 −0.67 . . . 0.95 2.95 Thr 4 A . . . . . . 1.86−0.67 . . . 0.95 2.95 Gln 5 A . . . . . . 1.66 −1.29 . . F 1.10 2.95 Gln6 A . . . . . . 2.04 −0.79 . . F 1.10 1.72 Gly 7 . . . . . . C 2.16−0.36 . . F 1.00 1.84 Glu 8 . . . . . . C 1.81 −0.84 . . F 1.64 2.08 Ala9 . . . . . T C 2.12 −0.81 . * F 2.18 1.19 Pro 10 . . . . . T C 1.31−0.81 * * F 2.52 2.08 Arg 11 . . . . T T . 1.42 −0.56 * * F 2.91 0.99Gly 12 . . . . T T . 1.42 −0.56 * * F 3.40 1.92 Gln 13 . . . . . . C1.42 −0.63 * * F 2.66 1.23 Leu 14 . . . . . . C 1.71 −1.06 * * F 2.551.09 Arg 15 . . . . . . C 1.33 −0.67 * * F 2.44 1.47 Gly 16 . . . . . .C 0.63 −0.60 * * F 2.18 0.86 Glu 17 . . . . T . . 0.77 −0.50 . * F 2.121.05 Ser 18 . . . . . . C −0.09 −0.76 . * F 2.30 0.83 Ala 19 . . . . . .C 0.51 −0.11 . * . 1.62 0.62 Ala 20 . . . . . . C 0.40 −0.11 . * . 1.390.56 Pro 21 . . . . . . C 0.16 0.29 * . . 0.56 0.72 Val 22 A . . . . . .−0.66 0.40 * . . −0.17 0.72 Pro 23 A . . . . . . −1.17 0.59 . . . −0.400.59 Gln 24 A . . B . . . −1.39 0.77 . . . −0.60 0.31 Ala 25 A . . B . .. −1.66 1.03 . . . −0.60 0.35 Leu 26 A . . B . . . −2.26 1.03 . . .−0.60 0.17 Leu 27 A . . B . . . −2.21 1.29 . . . −0.60 0.08 Leu 28 A . .B . . . −2.34 1.57 . . . −0.60 0.06 Val 29 A . . B . . . −2.93 1.50 * *. −0.60 0.08 Leu 30 A . . B . . . −2.23 1.31 . * . −0.60 0.10 Leu 31 A .. B . . . −2.01 0.63 . * . −0.60 0.23 Gly 32 A . . B . . . −1.20 0.44. * . −0.60 0.31 Ala 33 A . . . . . . −0.73 0.20 . * . 0.24 0.65 Arg 34A . . . . . . −0.22 −0.06 . * . 1.18 0.78 Ala 35 A . . . . T . 0.28−0.31 * * F 1.87 0.78 Gln 36 . . . . T T . 1.20 −0.26 * * F 2.76 1.11Gly 37 . . . . T T . 1.24 −0.76 * * F 3.40 1.11 Gly 38 . . . . T T .1.62 −0.37 * * F 2.76 1.47 Thr 39 . . . . T . . 1.62 −0.44 * * F 2.531.32 Arg 40 . . . . T . . 1.54 −0.84 * * F 2.80 2.60 Ser 41 . . . . . TC 1.54 −0.70 * * F 2.77 1.41 Pro 42 . . . . T T . 1.22 −1.13 * . F 2.941.63 Arg 43 . . . . T T . 0.98 −1.04 * . F 3.10 0.45 Cys 44 . . . . T T. 0.94 −0.54 . * . 2.64 0.34 Asp 45 . . . . T . . 0.83 −0.50 . * . 1.830.22 Cys 46 A . . . . T . 0.43 −0.93 . * . 1.62 0.18 Ala 47 A . . . . T. 0.61 −0.14 . * . 1.01 0.30 Gly 48 A . . . . T . 0.54 −0.21 * * . 0.700.24 Asp 49 A . . . . T . 1.26 −0.21 * * . 0.70 0.90 Phe 50 A . . . . .. 0.37 −0.79 * * F 1.10 1.79 His 51 A . . . . . . 0.69 −0.60 * * F 1.101.26 Lys 52 A . . . . . . 0.47 −0.60 * * F 0.95 0.75 Lys 53 . . . B T .. 0.11 0.09 * * F 0.25 0.71 Ile 54 . . . B T . . −0.56 0.09 * * . 0.100.45 Gly 55 . . . B T . . −0.52 0.16 * * . 0.10 0.12 Leu 56 . . . B T .. −0.38 0.73 * * . −0.20 0.03 Phe 57 . . . B T . . −0.77 0.73 * . .−0.20 0.09 Cys 58 . . . B T . . −1.48 0.47 * * . −0.20 0.09 Cys 59 . . .. T T . −0.80 0.61 * . . 0.42 0.06 Arg 60 . . . . T T . −1.04 0.36 . * .0.94 0.11 Gly 61 . . . . T T . −0.58 0.07 . * . 1.16 0.20 Cys 62 . . . .T T . 0.09 −0.07 * * . 1.98 0.37 Pro 63 . . . . T T . 0.51 −0.14 * * .2.20 0.26 Ala 64 . . . . T T . 0.37 0.61 . * . 1.08 0.41 Gly 65 . . . .T T . 0.30 0.87 . * . 0.86 0.62 His 66 . . . . T T . 0.06 0.30 * . .0.94 0.81 Tyr 67 . . . . T . . 0.51 0.37 . * . 0.52 0.81 Leu 68 . . . .T . . 0.06 0.30 * * . 0.76 1.26 Lys 69 . . . . T . . 0.33 0.44 * . .0.62 0.50 Ala 70 . . . . . T C 0.68 0.43 . . . 0.93 0.46 Pro 71 . . . .T T . 0.50 −0.33 . . F 2.49 0.96 Cys 72 . . . . T T . 0.08 −0.59 . * F3.10 0.74 Thr 73 . . . . T T . 0.54 −0.01 . * F 2.49 0.39 Glu 74 . . . .. T C 0.50 −0.09 . . F 2.11 0.25 Pro 75 . . . . T T . 0.79 −0.11 . . F2.13 0.76 Cys 76 . . . . T T . 0.69 −0.30 . . F 1.95 0.70 Gly 77 . . . .T T . 0.69 −0.30 . . F 1.77 0.58 Asn 78 . . . . T T . 0.19 0.27 . . F1.30 0.20 Ser 79 . . . . T T . −0.67 0.53 . . F 0.87 0.31 Thr 80 . . . .T T . −1.12 0.60 . . F 0.74 0.23 Cys 81 . . . . T T . −0.67 0.74 . . .0.46 0.08 Leu 82 . . B B . . . −0.32 0.77 . . . −0.47 0.09 Val 83 . . BB . . . −0.32 0.79 . . . −0.60 0.11 Cys 84 . . B B . . . −0.33 0.30 . .. −0.30 0.34 Pro 85 . . . . T T . −0.72 0.21 . . F 0.65 0.59 Gln 86 . .. . T T . −0.87 0.31 . . F 0.65 0.69 Asp 87 A . . . . T . −0.64 0.36 . .F 0.40 1.06 Thr 88 A . . . . T . −0.08 0.29 . . F 0.25 0.69 Phe 89 A A .. . . . 0.59 0.77 . . . −0.60 0.42 Leu 90 A A . . . . . 0.80 0.37 . . .−0.30 0.43 Ala 91 A A . . . . . 0.77 0.77 . . . −0.60 0.48 Trp 92 A A .. . . . 0.73 0.79 . . . −0.60 0.76 Glu 93 A A . . . . . 1.04 0.50 . . .−0.45 1.26 Asn 94 A A . . . . . 1.44 0.21 . . . −0.15 2.00 His 95 . A .. T . . 2.26 0.10 . . . 0.56 2.55 His 96 . A . . T . . 2.18 −0.81 . . F1.92 2.55 Asn 97 . . . . T T . 1.88 −0.24 . * F 2.18 0.85 Ser 98 . . . .T T . 1.99 −0.14 . . F 2.49 0.63 Glu 99 . . . . T T . 1.32 −0.64 . * F3.10 0.91 Cys 100 . . . . T T . 1.36 −0.57 . * . 2.64 0.30 Ala 101 A A .. . . . 0.80 −0.57 . * . 1.53 0.39 Arg 102 A A . . . . . 0.13 −0.46 . *. 0.92 0.23 Cys 103 A A . . . . . 0.43 0.11 . * . 0.01 0.23 Gln 104 A A. . . . . 0.43 −0.46 . * . 0.30 0.38 Ala 105 A A . . . . . 1.10 −0.96. * . 0.60 0.33 Cys 106 A A . . . . . 1.10 −0.56 . * . 0.75 1.08 Asp 107A A . . . . . 0.69 −0.63 . * F 0.75 0.63 Glu 108 A A . . . . . 1.36−0.64 * . F 0.75 0.83 Gln 109 A A . . . . . 0.50 −0.74 * . F 0.90 2.69Ala 110 A A . . . . . 0.50 −0.67 . . F 0.90 1.20 Ser 111 A A . . . . .0.36 −0.17 * . F 0.45 0.70 Gln 112 A A . . . . . 0.36 0.51 . . . −0.600.33 Val 113 A A . . . . . 0.36 0.11 * . . −0.30 0.57 Ala 114 A A . . .. . −0.31 0.01 * . . −0.30 0.68 Leu 115 A A . . . . . −0.02 0.20 . . .−0.30 0.21 Glu 116 A A . . . . . −0.31 0.19 . . . −0.30 0.38 Asn 117 A A. . . . . −1.17 0.04 . . . −0.30 0.38 Cys 118 A A . . . . . −0.90 0.19 *. . −0.30 0.34 Ser 119 A A . . . . . −0.31 −0.00 * . . 0.30 0.20 Ala 120A . . . . . . 0.19 −0.00 . * . 0.50 0.21 Val 121 A . . . . . . 0.30 0.09. * . −0.10 0.56 Ala 122 A . . . . . . −0.37 −0.49 . * . 0.78 0.82 Asp123 A . . . . T . −0.04 −0.30 . * F 1.41 0.43 Thr 124 . . . . T T .−0.41 −0.37 * * F 2.09 0.58 Arg 125 . . . . T T . 0.22 −0.44 * * F 2.370.31 Cys 126 . . . . T T . 0.87 −0.94 * * . 2.80 0.37 Gly 127 . . . . T. . 1.11 −0.51 * * . 2.32 0.39 Cys 128 . . . . T . . 0.82 −0.57 * * .2.04 0.20 Lys 129 . . . . . T C 0.43 0.34 * * F 1.01 0.39 Pro 130 . . .. T T . −0.53 0.56 * * F 0.63 0.34 Gly 131 . . . . T T . 0.13 0.77 . . .0.20 0.47 Trp 132 . . . . T T . −0.19 0.20 . * . 0.50 0.41 Phe 133 A . .B . . . 0.48 0.77 . * . −0.60 0.14 Val 134 A . . B . . . −0.42 0.74 . *. −0.60 0.25 Glu 135 A . . B . . . −0.51 0.96 . * . −0.60 0.18 Cys 136 .. . B T . . −0.17 0.43 . . . −0.20 0.27 Gln 137 . . . B T . . −0.54 0.04. * . 0.10 0.63 Val 138 . . . B T . . −0.70 −0.03 . * . 0.70 0.20 Ser139 . . . B T . . −0.14 0.61 * * . −0.20 0.27 Gln 140 . . . B T . .−0.44 0.43 * * . −0.20 0.21 Cys 141 . . . B T . . −0.08 0.41 * . . −0.200.38 Val 142 . . . B T . . −0.29 0.16 . . F 0.25 0.38 Ser 143 . . . . T. . −0.13 0.20 . . F 0.45 0.34 Ser 144 . . . . T . . −0.08 0.59 . . F0.15 0.55 Ser 145 . . . . . T C −0.74 0.77 . . F 0.30 1.16 Pro 146 . . .. T T . −0.08 0.70 . . F 0.35 0.46 Phe 147 . . . . T T . 0.57 0.71 . . .0.20 0.60 Tyr 148 . . . . T T . 0.20 0.76 . . . 0.20 0.69 Cys 149 . . .. T . . −0.31 0.94 . * . 0.00 0.24 Gln 150 . . B . . T . −0.01 1.20 . *. −0.20 0.23 Pro 151 . . . . T T . −0.47 0.41 . * . 0.20 0.24 Cys 152 .. . . T T . −0.11 0.23 . * . 0.50 0.24 Leu 153 . . . . T T . −0.46 0.09. . . 0.50 0.14 Asp 154 . . . . T T . −0.60 0.19 . * . 0.50 0.09 Cys 155A . . . . T . −0.63 0.44 . * . −0.20 0.14 Gly 156 A . . . . T . −0.310.37 . . . 0.10 0.23 Ala 157 A . . . . T . 0.32 −0.31 . . . 0.70 0.27Leu 158 A A . . . . . 0.82 0.19 * * . −0.30 0.69 His 159 A A . . . . .0.93 0.10 * * . −0.30 1.00 Arg 160 A A . . . . . 0.79 −0.33 * . . 0.451.94 His 161 A A . . . . . 0.32 −0.14 * . . 0.45 1.94 Thr 162 A A . . .. . 0.24 −0.14 * . . 0.45 1.17 Arg 163 . A . . T . . 0.76 −0.07 * . .0.70 0.32 Leu 164 . A . . T . . 0.90 0.31 . . . 0.44 0.32 Leu 165 . A .. T . . 0.90 −0.19 . . . 1.38 0.43 Cys 166 . . . . T T . 0.93 −0.67 . *. 2.42 0.43 Ser 167 . . . . T T . 0.93 −0.67 . * . 2.76 0.87 Arg 168 . .. . T T . 0.82 −0.87 . * F 3.40 1.52 Arg 169 . . . . T T . 0.97 −1.56 *. F 3.06 4.74 Asp 170 . . . . T T . 1.43 −1.56 * . F 2.81 1.90 Thr 171 .. . . T T . 1.79 −1.51 * . F 2.41 0.96 Asp 172 . . . . T T . 1.42−1.03 * . F 2.16 0.71 Cys 173 . . . . T T . 0.50 −0.46 * . F 1.61 0.23Gly 174 . . . . T . . 0.18 0.23 * . F 0.90 0.13 Thr 175 . . . . T . .−0.17 0.17 . . . 0.66 0.12 Cys 176 . . . . . . C −0.56 0.60 * . . 0.070.22 Leu 177 . . . . . T C −0.80 0.81 * . . 0.18 0.19 Pro 178 . . . . .T C −0.13 1.14 * . . 0.09 0.21 Gly 179 . . . . T T . 0.18 0.66 * . .0.45 0.68 Phe 180 . . . . T T . 0.14 0.59 * . . 0.85 1.12 Tyr 181 . . .. T . . 0.81 0.33 * . . 1.05 0.72 Glu 182 . . . . T . . 1.28 −0.10 * . .2.05 1.21 His 183 . . . . T T . 0.82 −0.10 * * . 2.50 1.38 Gly 184 . . .. T T . 0.31 −0.31 * * . 2.10 0.47 Asp 185 . . . . T T . 0.71 −0.43 * *. 1.85 0.20 Gly 186 . . . . T T . 0.29 −0.04 . . . 1.60 0.20 Cys 187 . .. . T . . 0.08 0.03 . * . 0.55 0.11 Val 188 . . . . T . . −0.20 0.03 . *. 0.30 0.10 Ser 189 . . . . T . . −0.16 0.51 . * . 0.00 0.15 Cys 190 . .B . . T . −0.47 0.47 . . . −0.20 0.37 Pro 191 . . . . T T . −0.93 0.39 .. F 0.65 0.71 Thr 192 . . . . T T . −0.61 0.43 . . F 0.35 0.44 Ser 193 .. . . T T . −0.06 0.47 . . F 0.35 0.81 Thr 194 . . . . T . . −0.42 0.29. . F 0.45 0.70 Leu 195 . . . . T . . 0.03 0.43 . . F 0.46 0.26 Gly 196. . . . T . . 0.24 0.37 * . F 1.07 0.30 Ser 197 . . . . T . . 0.67−0.01 * . F 1.98 0.36 Cys 198 . . . . . T C 0.30 −0.50 * . F 2.59 0.85Pro 199 . . . . T T . 0.02 −0.61 * . F 3.10 0.46 Glu 200 . . . . T T .0.24 −0.54 * . F 2.79 0.35 Arg 201 A . . . . T . −0.27 −0.43 * . . 1.630.66 Cys 202 A . . B . . . −0.63 −0.36 * . . 0.92 0.32 Ala 203 A . . B .. . −0.31 −0.21 . . . 0.61 0.10 Ala 204 A . . B . . . −0.39 0.21 * * .−0.30 0.05 Val 205 A . . B . . . −0.28 1.13 . . . −0.60 0.10 Cys 206 A .. B . . . −0.39 0.56 * * . −0.60 0.19 Gly 207 . . . B T . . −0.32 0.46 *. . −0.20 0.32 Trp 208 . . . B T . . −0.43 0.57 * . . −0.20 0.43 Arg 209A . . B . . . −0.13 0.71 * . . −0.60 0.69 Gln 210 A . . B . . . −0.131.06 . * . −0.60 0.74 Met 211 A . . B . . . 0.53 1.27 . * . −0.60 0.52Phe 212 . . . B T . . 0.02 0.76 . * . −0.20 0.46 Trp 213 A . . B . . .−0.50 1.40 . * . −0.60 0.20 Val 214 A . . B . . . −1.42 1.69 . * . −0.600.16 Gln 215 A . . B . . . −2.01 1.76 . . . −0.60 0.16 Val 216 A . . B .. . −1.76 1.47 . . . −0.60 0.15 Leu 217 A . . B . . . −1.87 0.99 . * .−0.60 0.20 Leu 218 A . . B . . . −2.43 1.03 . . . −0.60 0.10 Ala 219 A .. B . . . −2.43 1.27 . . . −0.60 0.10 Gly 220 A . . B . . . −2.64 1.27 .. . −0.60 0.09 Leu 221 A . . B . . . −2.60 1.01 . . . −0.60 0.16 Val 222. . B B . . . −2.60 1.01 . . . −0.60 0.13 Val 223 . . B B . . . −2.601.20 . . . −0.60 0.11 Pro 224 . . B B . . . −2.36 1.46 . . . −0.60 0.11Leu 225 . . B B . . . −2.60 1.20 . . . −0.60 0.15 Leu 226 A . . B . . .−2.10 1.06 . * . −0.60 0.20 Leu 227 A . . B . . . −2.06 0.90 . . . −0.600.19 Gly 228 A . . B . . . −1.51 1.16 . * . −0.60 0.19 Ala 229 A . . B .. . −1.54 0.96 . . . −0.60 0.32 Thr 230 A . . B . . . −1.04 1.03 . * .−0.60 0.62 Leu 231 A . . B . . . −0.48 0.83 * * . −0.60 0.90 Thr 232 . .B B . . . 0.44 1.16 * * . −0.45 1.40 Tyr 233 . . . B T . . 0.76 0.66 * *. −0.05 1.89 Thr 234 . . . B T . . 0.68 0.67 * * . −0.05 3.12 Tyr 235 .. . . T T . 0.70 0.56 * * . 0.35 1.16 Arg 236 . . . . T T . 1.300.99 * * . 0.20 0.78 His 237 . . . . T T . 1.58 0.66 . * . 0.20 0.83 Cys238 . . . . T T . 1.87 0.67 . * . 0.20 0.72 Trp 239 . . . . . T C 1.97−0.09 . * . 0.90 0.74 Pro 240 . . . . T T . 1.40 0.34 . * . 0.50 0.84His 241 . . . . T T . 0.43 0.53 . * . 0.35 1.29 Lys 242 . . . . . T C0.16 0.60 . . F 0.15 0.91 Pro 243 . . . . . . C 0.23 0.17 . * F 0.250.85 Leu 244 . A . . . . C 0.52 0.24 * . . −0.10 0.63 Val 245 A A . . .. . 0.73 −0.26 * . . 0.30 0.53 Thr 246 A A . . . . . 0.18 −0.26 * . .0.30 0.59 Ala 247 A A . . . . . −0.21 −0.19 * . F 0.45 0.72 Asp 248 A A. . . . . −0.60 −0.44 . . F 0.45 0.97 Glu 249 A A . . . . . 0.21 −0.47 .. F 0.45 0.66 Ala 250 A A . . . . . 0.48 −0.96 . . F 0.90 1.14 Gly 251 AA . . . . . −0.02 −0.96 * . . 0.60 0.69 Met 252 A A . . . . . 0.26−0.27 * . . 0.30 0.33 Glu 253 A A . . . . . 0.04 0.21 . . . −0.30 0.47Ala 254 A A . . . . . −0.17 0.14 * . . −0.30 0.73 Leu 255 . A . . . . C0.21 0.14 . . . 0.05 1.14 Thr 256 . A . . . . C −0.03 −0.04 . . F 0.801.02 Pro 257 . A . . . . C 0.26 0.46 . . F −0.10 1.02 Pro 258 . . . . .T C 0.22 0.44 . . F 0.30 1.78 Pro 259 . . . . T T . −0.00 0.26 . . F0.80 1.68 Ala 260 . . . . T T . 0.51 0.46 . . F 0.35 0.90 Thr 261 A . .. . T . 0.61 0.41 . . . −0.20 0.78 His 262 . . B . . . . 0.01 0.41 . . .−0.40 0.78 Leu 263 . . B . . . . 0.22 0.67 . . . −0.40 0.63 Ser 264 . .. . . T C 0.13 0.17 . . F 0.45 0.73 Pro 265 . . . . . T C 0.13 0.07 . .F 0.45 0.72 Leu 266 . . . . . T C 0.41 0.07 . . F 0.45 0.89 Asp 267 A .. . . T . 0.13 −0.11 . . F 0.85 0.90 Ser 268 A A . . . . . 0.13 −0.01 *. F 0.45 0.84 Ala 269 A A . . . . . −0.38 0.24 * . . −0.30 0.84 His 270A A . . . . . −0.76 0.24 * . . −0.30 0.41 Thr 271 . A B . . . . −0.160.74 * . . −0.60 0.31 Leu 272 . A B . . . . −0.37 0.79 * . . −0.60 0.48Leu 273 . A B . . . . −0.07 0.71 . . . −0.26 0.54 Ala 274 . A . . . . C0.22 0.21 . . . 0.58 0.63 Pro 275 . . . . . T C −0.04 0.11 . . F 1.621.02 Pro 276 . . . . . T C 0.27 −0.19 . . F 2.56 1.66 Asp 277 . . . . TT . 1.12 −0.87 * * F 3.40 2.85 Ser 278 A . . . . T . 1.04 −1.37 . * F2.66 3.68 Ser 279 A . . . . . . 0.97 −1.11 * * F 2.12 1.67 Glu 280 A . .. . . . 0.87 −0.97 * . F 1.63 0.54 Lys 281 A . . B . . . 0.22 −0.49 * .F 0.79 0.58 Ile 282 A . . B . . . 0.22 −0.23 . * . 0.30 0.32 Cys 283 A .. B . . . −0.29 −0.21 . . . 0.30 0.32 Thr 284 . . B B . . . −0.84 0.47 .. . −0.60 0.13 Val 285 . . B B . . . −1.19 1.11 . . . −0.60 0.14 Gln 286. . B B . . . −1.23 0.86 . . . −0.60 0.26 Leu 287 . . B B . . . −0.640.69 * . . −0.60 0.29 Val 288 . . . B T . . −0.27 0.59 * * . −0.20 0.52Gly 289 . . . . T T . −0.27 0.86 * . F 0.35 0.31 Asn 290 . . . . T T .0.38 0.94 * . F 0.35 0.55 Ser 291 . . . . T T . 0.03 0.69 * . F 0.501.15 Trp 292 . . . . . T C 0.60 0.47 . . F 0.30 1.15 Thr 293 . . . . . TC 1.24 0.80 * . F 0.30 1.12 Pro 294 . . . . . T C 1.59 0.83 . . F 0.301.29 Gly 295 . . . . . T C 1.28 0.44 . . F 0.30 2.12 Tyr 296 . . . . . TC 1.58 0.01 . . F 0.60 2.12 Pro 297 . . . . . . C 1.87 −0.07 . . F 1.002.38 Glu 298 . A . . T . . 1.59 −0.50 . . F 1.30 4.16 Thr 299 A A . . .. . 0.99 −0.43 . . F 0.60 2.68 Gln 300 A A . . . . . 0.67 −0.50 . . F0.90 1.43 Glu 301 A A . . . . . 0.70 −0.36 . . F 0.45 0.44 Ala 302 A A .. . . . 0.91 0.07 . . . −0.30 0.47 Leu 303 A A . . . . . 0.06 −0.01 . .. 0.30 0.47 Cys 304 A A . B . . . 0.06 0.23 . * . −0.30 0.20 Pro 305 . A. B T . . −0.23 0.71 . * . −0.20 0.29 Gln 306 . . . B T . . −0.53 1.13. * . −0.20 0.37 Val 307 . . . B T . . −0.23 0.83 . * . −0.20 0.93 Thr308 . . . B T . . 0.58 1.17 * * . −0.20 0.63 Trp 309 . . . B T . . 1.240.74 * * . −0.20 0.61 Ser 310 . . . B T . . 0.64 0.74 * * . −0.05 1.42Trp 311 . . . B T . . 0.43 0.79 * * . 0.10 0.81 Asp 312 . . . B T . .0.99 0.73 * * F 0.70 1.19 Gln 313 . . . . . . C 1.41 0.20 * * F 1.301.19 Leu 314 . . . . . T C 1.11 −0.19 * . F 2.40 2.22 Pro 315 . . . . .T C 0.60 −0.60 * * F 3.00 1.34 Ser 316 . . . . T T . 0.54 0.09 * * F1.85 0.64 Arg 317 . . . . T T . 0.33 0.11 * * F 1.55 0.77 Ala 318 . . .. T . . −0.26 −0.14 * * F 1.65 0.77 Leu 319 . . . . . . C −0.03−0.07 * * F 1.15 0.58 Gly 320 . . . . . . C −0.41 0.04 * . F 0.25 0.30Pro 321 . A . . . . C −0.32 0.54 * . . −0.40 0.30 Ala 322 . A . . . . C−0.74 0.47 * * . −0.40 0.56 Ala 323 A A . . . . . −0.97 0.27 . . . −0.300.82 Ala 324 . A . . . . C −0.46 0.53 . . . −0.40 0.43 Pro 325 . . . . .. C −0.32 0.49 . . F −0.05 0.58 Thr 326 . . . . . . C −0.11 0.41 . . F−0.05 0.88 Leu 327 . . . . . . C 0.18 −0.09 . . F 1.00 1.51 Ser 328 . .. . . T C 0.56 −0.20 . . F 1.20 1.31 Pro 329 . . . . . T C 0.56 −0.20 .. F 1.45 1.41 Glu 330 . . . . . T C 0.42 −0.19 . . F 1.70 1.72 Ser 331 .. . . . T C 0.43 −0.44 . . F 1.95 1.27 Pro 332 . . . . . T C 1.03 −0.44. . F 2.20 1.10 Ala 333 . . . . T T . 0.74 −0.44 . . F 2.50 0.98 Gly 334. . . . . T C 0.36 0.06 . . F 1.45 0.74 Ser 335 . . . . . T C −0.24 0.29. . F 1.20 0.47 Pro 336 A A . . . . . −0.76 0.47 . . F 0.05 0.47 Ala 337A A . . . . . −0.54 0.66 . . . −0.35 0.39 Met 338 . A B . . . . −0.170.63 . . . −0.60 0.50 Met 339 . A B . . . . −0.17 0.67 . . . −0.60 0.50Leu 340 . A B . . . . −0.08 0.67 . . . −0.60 0.49 Gln 341 . . . . . T C0.13 0.60 * * . 0.00 0.77 Pro 342 . . . . . T C −0.09 0.39 * * F 0.601.34 Gly 343 . . . . . T C 0.27 0.46 * * F 0.30 1.34 Pro 344 . . . . . TC 0.87 0.53 * . F 0.30 1.21 Gln 345 . A . . . . C 0.82 0.13 * . F 0.201.31 Leu 346 . A B . . . . 0.22 0.34 * . . −0.30 0.98 Tyr 347 . A B . .. . 0.43 0.53 * . . −0.60 0.63 Asp 348 . A B . . . . 0.19 0.10 * . .−0.30 0.61 Val 349 . A B . . . . −0.46 0.20 * . . −0.30 0.74 Met 350 . AB . . . . −0.67 0.16 * . . −0.30 0.35 Asp 351 A A . . . . . −0.44−0.17 * * . 0.30 0.33 Ala 352 A A . . . . . −0.09 0.33 . . . −0.30 0.44Val 353 A A . . . . . 0.02 −0.31 . . . 0.30 0.88 Pro 354 A . . . . . .0.59 −0.93 . . . 0.95 1.03 Ala 355 A A . . . . . 1.23 −0.01 * . . 0.451.07 Arg 356 A A . . . . . 1.23 −0.51 * . F 0.90 2.89 Arg 357 A A . . .. . 1.12 −1.16 * . F 0.90 3.24 Trp 358 A A . . . . . 1.12 −0.80 * * F0.90 2.77 Lys 359 A A . . . . . 1.44 −0.66 * * F 0.90 1.05 Glu 360 A A .. . . . 1.72 −0.66 * * . 0.75 1.05 Phe 361 A A . . . . . 0.80 −0.17 * *. 0.45 1.44 Val 362 A A . . . . . 0.34 −0.40 * * . 0.30 0.59 Arg 363 A A. . . . . −0.18 0.03 * * . −0.30 0.34 Thr 364 A A . . . . . −0.11 0.71 *. . −0.60 0.32 Leu 365 A A . . . . C −0.11 −0.07 * . . 0.50 0.85 Gly 366A A . . . . . −0.00 −0.71 * . . 0.60 0.76 Leu 367 A A . . . . . 0.86−0.21 * . . 0.30 0.53 Arg 368 A A . . . . . −0.14 −0.70 . . . 0.75 1.11Glu 369 A A . . . . . 0.17 −0.70 . * F 0.75 0.79 Ala 370 A A . . . . .0.39 −1.13 . . F 0.90 1.65 Glu 371 A A . . . . . −0.12 −1.31 * . . 0.600.85 Ile 372 A A . . . . . 0.69 −0.67 * . . 0.60 0.37 Glu 373 A A . . .. . −0.28 −0.67 . . . 0.60 0.63 Ala 374 A A . . . . . −0.28 −0.53 . * .0.60 0.27 Val 375 A A . . . . . −0.58 −0.53 . * . 0.60 0.66 Glu 376 A A. . . . . −0.92 −0.53 * . . 0.60 0.27 Val 377 A A . . . . . 0.08 −0.10 *. . 0.30 0.26 Glu 378 A A . . . . . −0.62 −0.60 * * . 0.60 0.69 Ile 379A A . . . . . 0.08 −0.46 * * . 0.30 0.35 Gly 380 A A . . . . . 0.93−0.46 * * . 0.30 0.91 Arg 381 A A . . . . . 0.93 −1.10 . * F 0.75 0.88Phe 382 A A . . . . . 1.79 −0.70 . * F 0.90 2.18 Arg 383 A A . . . . .1.54 −0.99 * * F 0.90 3.81 Asp 384 A A . . . . . 2.43 −0.66 * * F 0.903.05 Gln 385 A A . . . . . 2.18 −0.66 * * F 0.90 6.10 Gln 386 A A . . .. . 1.26 −0.83 * * F 0.90 3.08 Tyr 387 A A . . . . . 2.00 −0.14 * * .0.45 1.52 Glu 388 A A . . . . . 2.00 −0.14 * * . 0.45 1.76 Met 389 A A .. . . . 1.71 −0.54 * * . 0.75 1.99 Leu 390 A A . . . . . 1.82 −0.03 * *. 0.45 1.33 Lys 391 . A . . T . . 1.82 −0.79 * * . 1.15 1.51 Arg 392 . A. . T . . 2.07 −0.39 * * F 1.00 2.64 Trp 393 . A . . T . . 2.07−0.60 * * F 1.30 5.55 Arg 394 A A . . . . . 2.46 −0.89 * * F 0.90 4.80Gln 395 . A . . T . . 2.68 −0.46 * * F 1.00 3.79 Gln 396 . A . . . . C2.29 0.04 * * F 0.20 3.64 Gln 397 . . . . . T C 1.37 −0.44 . * F 1.201.84 Pro 398 . . . . . T C 1.31 0.24 . * F 0.45 0.88 Ala 399 . . . . T T. 0.61 0.27 . . F 0.65 0.50 Gly 400 . . . . . T C −0.24 0.37 . . . 0.300.29 Leu 401 . . . . . . C −0.49 0.61 . . . −0.20 0.14 Gly 402 . . . . .. C −1.08 0.94 . . . −0.20 0.22 Ala 403 A A . . . . . −1.46 0.94 . . .−0.60 0.22 Val 404 A A . . . . . −1.68 1.01 . . . −0.60 0.27 Tyr 405 A A. . . . . −1.33 1.01 * . . −0.60 0.23 Ala 406 A A . . . . . −0.41 0.59 *. . −0.60 0.39 Ala 407 A A . . . . . −0.67 0.09 * . . −0.15 1.03 Leu 408A A . . . . . −0.42 0.06 * . . −0.30 0.65 Glu 409 A A . . . . . −0.38−0.27 * * . 0.30 0.63 Arg 410 A A . . . . . −0.13 −0.09 * * . 0.30 0.52Met 411 A A . . . . . 0.11 −0.59 * * . 0.75 1.05 Gly 412 A . . . . . .0.03 −0.84 * * . 0.80 0.60 Leu 413 A . . . . T . −0.01 −0.27 * * . 0.700.16 Asp 414 A . . . . T . −0.01 0.37 . * . 0.10 0.12 Gly 415 A . . . .T . −0.12 −0.24 * * . 0.70 0.22 Cys 416 A . . . . T . −0.33 −0.67 * * .1.00 0.44 Val 417 A A . . . . . 0.12 −0.67 * * . 0.60 0.22 Glu 418 A A .. . . . 0.63 −0.67 * * . 0.60 0.43 Asp 419 A A . . . . . 0.74 −0.71 * *F 0.90 1.07 Leu 420 A A . . . . . 0.28 −1.29 * * F 0.90 2.81 Arg 421 A A. . . . . 0.94 −1.24 * * F 0.90 1.34 Ser 422 A A . . . . . 1.91−0.84 * * F 0.90 1.39 Arg 423 . A . . T . . 1.57 −0.84 * * F 1.30 3.30Leu 424 . A . . T . . 1.36 −1.10 * * F 1.30 1.67 Gln 425 . . . . T T .1.78 −0.67 * * F 1.70 1.92 Arg 426 . . . . T T . 1.28 −0.63 * * . 1.551.25 Gly 427 . . . . . T C 1.19 −0.20 * . . 1.05 1.94 Pro 428 . . . . .T C 0.69 −0.46 * . . 1.05 1.44

[0180] Among highly preferred fragments in this regard are those thatcomprise, or alternatively consist of, regions of DR3-V1 and DR3 thatcombine several structural features, such as several of the features setout above in Table 2.

[0181] The invention further provides for the proteins containingpolypeptide sequences encoded by the polynucleotides of the invention.

[0182] The present invention is further directed to isolatedpolypeptides comprising, or alternatively consisting of, fragments ofDR3-V1 and DR3. In particular, the invention provides isolatedpolypeptides comprising, or alternatively consisting of, the amino acidsequences of a member selected from the group consisting of amino acids1-60, 11-70, 21-80, 31-90, 41-100, 51-110, 61-120, 71-130, 81-140,91-150, 101-160, 111-170, 121-180, 131-190, 141-200, 151-210, 161-220,171-230, 181-240, 191-250, 201-260, 211-270, 221-280, 231-290, 241-300,251-310, 261-320, 271-330, 281-340, 291-350, 301-360, 311-370, 321-380,331-390, 341-400, 351-410, 361-420, and 371-428 of SEQ ID NO:2, as wellas isolated polynucleotides which encode these polypeptides. Theinvention also provides isolated polypeptides comprising, oralternatively consisting of, the amino acid sequences of a memberselected from the group consisting of amino acids 1-60, 11-70, 21-80,31-90, 41-100, 51-110, 61-120, 71-130, 81-140, 91-150, 101-160, 111-170,121-180, 131-190, 141-200, 151-210, 161-220, 171-230, 181-240, 191-250,201-260, 211-270, 221-280, 231-290, 241-300, 251-310, 261-320, 271-330,281-340, 291-350, 301-360, 311-370, 321-380, 331-390, 341-400, 351-410,and 361-417 of SEQ ID NO:4, as well as isolated polynucleotides whichencode these polypeptides.

[0183] The DR3-V1 or DR3 proteins of the invention may be in monomers ormultimers (i.e., dimers, trimers, tetramers, and higher multimers).Accordingly, the present invention relates to monomers and multimers ofthe DR3-V1 or DR3 proteins of the invention, their preparation, andcompositions (preferably, pharmaceutical compositions) containing them.In specific embodiments, the polypeptides of the invention are monomers,dimers, trimers or tetramers. In additional embodiments, the multimersof the invention are at least dimers, at least trimers, or at leasttetramers.

[0184] Multimers encompassed by the invention may be homomers orheteromers. As used herein, the term homomer, refers to a multimercontaining only DR3-V1 or DR3 proteins of the invention (includingDR3-V1 or DR3 fragments, variants, and fusion proteins, as describedherein). These homomers may contain DR3-V1 or DR3 proteins havingidentical or different polypeptide sequences. In a specific embodiment,a homomer of the invention is a multimer containing only DR3-V1 or DR3proteins having an identical polypeptide sequence. In another specificembodiment, a homomer of the invention is a multimer containing DR3-V1or DR3 proteins having different polypeptide sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing DR3-V1 or DR3 proteins having identical or differentpolypeptide sequences) or a homotrimer (e.g., containing DR3-V1 or DR3proteins having identical or different polypeptide sequences). Inadditional embodiments, the homomeric multimer of the invention is atleast a homodimer, at least a homotrimer, or at least a homotetramer.

[0185] As used herein, the term heteromer refers to a multimercontaining heterologous proteins (i.e., proteins containing onlypolypeptide sequences that do not correspond to a polypeptide sequencesencoded by the DR3 gene) in addition to the DR3-V1 or DR3 proteins ofthe invention. In a specific embodiment, the multimer of the inventionis a heterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the heteromeric multimer of the invention is at least aheterodimer, at least a heterotrimer, or at least a heterotetramer.

[0186] Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the DR3-V1 or DR3 proteins of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence of the protein (e.g., thepolypeptide sequence recited in SEQ ID NO:2 or the polypeptide encodedby the deposited cDNA). In one instance, the covalent associations arecross-linking between cysteine residues located within the polypeptidesequences of the proteins which interact in the native (i.e., naturallyoccurring) polypeptide. In another instance, the covalent associationsare the consequence of chemical or recombinant manipulation.Alternatively, such covalent associations may involve one or more aminoacid residues contained in the heterologous polypeptide sequence in aDR3-V1 or DR3 fusion protein. In one example, covalent associations arebetween the heterologous sequence contained in a fusion protein of theinvention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example,the covalent associations are between the heterologous sequencecontained in a DR3-V1-Fc or DR3-Fc fusion protein of the invention (asdescribed herein). In another specific example, covalent associations offusion proteins of the invention are between heterologous polypeptidesequences from another TNF family ligand/receptor member that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication No. WO 98/49305,the contents of which are herein incorporated by reference in itsentirety).

[0187] The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). Further, proteins of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide sequence of the protein and techniquesknown in the art may be applied to generate multimers containing one ormore of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925,which is herein incorporated by reference in its entirety).Additionally, techniques known in the art may be applied to generateliposomes containing the protein components desired to be contained inthe multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety).

[0188] Alternatively, multimers of the invention may be generated usinggenetic engineering techniques known in the art. In one embodiment,proteins contained in multimers of the invention are producedrecombinantly using fusion protein technology described herein orotherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety). In a specificembodiment, polynucleotides coding for a homodimer of the invention aregenerated by ligating a polynucleotide sequence encoding a polypeptideof the invention to a sequence encoding a linker polypeptide and thenfurther to a synthetic polynucleotide encoding the translated product ofthe polypeptide in the reverse orientation from the original C-terminusto the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat.No.5,478,925, which is herein incorporated by reference in itsentirety). In another embodiment, recombinant techniques describedherein or otherwise known in the art are applied to generate recombinantpolypeptides of the invention which contain a transmembrane domain andwhich can be incorporated by membrane reconstitution techniques intoliposomes (see, e.g., U.S. Pat. No.5,478,925, which is hereinincorporated by reference in its entirety).

[0189] Protein Modification

[0190] In addition, proteins of the invention can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., New York, and Hunkapiller, M. et al., Nature 310:105-111 (1984)).For example, a peptide corresponding to a fragment of the DR3-V1 or DR3polypeptides of the invention can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the DR3-V1 or DR3 polypeptide sequence. Non-classicalamino acids include, but are not limited to, to the D-isomers of thecommon amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid,4-aminobutyric acid, Abu, 2-amino butyric acid, α-Abu, α-Ahx, 6-aminohexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, α-alanine, fluoro-amino acids,designer amino acids such as α-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

[0191] Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

[0192] The invention additionally, encompasses DR3-V1 and DR3polypeptides which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to, specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction,metabolic synthesis in the presence of tunicamycin; etc.

[0193] Additional post-translational modifications encompassed by theinvention include, for example, e.g., N-linked or O-linked carbohydratechains, processing of N-terminal or C-terminal ends), attachment ofchemical moieties to the amino acid backbone, chemical modifications ofN-linked or O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

[0194] Also provided by the invention are chemically modifiedderivatives of DR3-V1 or DR3 which may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337).The chemical moieties for derivation may be selected from water solublepolymers such as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

[0195] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 1 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

[0196] As noted above, the polyethylene glycol may have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), thedisclosures of each of which are incorporated herein by reference.

[0197] The polyethylene glycol molecules (or other chemical moieties)should be attached to the protein with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384, herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues, glutamic acid residues and theC-terminal amino acid residue. Sulfhydryl groups may also be used as areactive group for attaching the polyethylene glycol molecules.Preferred for therapeutic purposes is attachment at an amino group, suchas attachment at the N-terminus or lysine group.

[0198] As suggested above, polyethylene glycol may be attached toproteins via linkage to any of a number of amino acid residues. Forexample, polyethylene glycol can be linked to a protein via covalentbonds to lysine, histidine, aspartic acid, glutamic acid, or cysteineresidues. One or more reaction chemistries may be employed to attachpolyethylene glycol to specific amino acid residues (e.g., lysine,histidine, aspartic acid, glutamic acid, or cysteine) of the protein orto more than one type of amino acid residue (e.g., lysine, histidine,aspartic acid, glutamic acid, cysteine and combinations thereof) of theprotein.

[0199] One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation, which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

[0200] As indicated above, pegylation of the proteins of the inventionmay be accomplished by any number of means. For example, polyethyleneglycol may be attached to the protein either directly or by anintervening linker. Linkerless systems for attaching polyethylene glycolto proteins are described in Delgado et al., Crit. Rev. Thera. DrugCarrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol.68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO95/06058; and WO 98/32466, the disclosures of each of which areincorporated herein by reference.

[0201] One system for attaching polyethylene glycol directly to aminoacid residues of proteins without an intervening linker employstresylated MPEG, which is produced by the modification of monmethoxypolyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Uponreaction of protein with tresylated MPEG, polyethylene glycol isdirectly attached to amine groups of the protein. Thus, the inventionincludes protein-polyethylene glycol conjugates produced by reactingproteins of the invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

[0202] Polyethylene glycol can also be attached to proteins using anumber of different intervening linkers. For example, U.S. Pat.No.5,612,460, the entire disclosure of which is incorporated herein byreference, discloses urethane linkers for connecting polyethylene glycolto proteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldimidazole,MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, andvarious MPEG-succinate derivatives. A number additional polyethyleneglycol derivatives and reaction chemistries for attaching polyethyleneglycol to proteins are described in WO 98/32466, the entire disclosureof which is incorporated herein by reference. Pegylated protein productsproduced using the reaction chemistries set out herein are includedwithin the scope of the invention.

[0203] The number of polyethylene glycol moieties attached to eachprotein of the invention (i.e., the degree of substitution) may alsovary. For example, the pegylated proteins of the invention may belinked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, ormore polyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

[0204] Polypeptide Assays

[0205] The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of DR3-V1 or DR3protein, or the soluble form thereof, in cells and tissues, includingdetermination of normal and abnormal levels. Thus, for instance, adiagnostic assay in accordance with the invention for detectingover-expression of DR3-V1 or DR3, or soluble form thereof, compared tonormal control tissue samples may be used to detect the presence oftumors, for example. Assay techniques that can be used to determinelevels of a protein, such as an DR3 protein of the present invention, ora soluble form thereof, in a sample derived from a host are well-knownto those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

[0206] Assaying DR3-V1 or DR3 protein levels in a biological sample canoccur using any art-known method. Preferred for assaying DR3-V1 or DR3protein levels in a biological sample are antibody-based techniques. Forexample, DR3-V1 or DR3 protein expression in tissues can be studied withclassical immunohistological methods. M. Jalkanen et al., J. Cell. Biol.101:976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 105:3087-3096(1987).

[0207] Other antibody-based methods useful for detecting DR3-V1 or DR3protein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA).

[0208] Suitable labels are known in the art and include enzyme labels,such as glucose oxidase, radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulphur (³⁵S), tritium (³H), indium (¹¹²In), andtechnetium (^(99m)Tc), and fluorescent labels, such as fluorescein andrhodamine, and biotin.

[0209] Antibodies

[0210] The present invention further relates to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,preferably an epitope, of the present invention (as determined byimmunoassays well known in the art for assaying specificantibody-antigen binding). Antibodies of the invention include, but arenot limited to, polyclonal, monoclonal, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

[0211] Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera V_(L) or V_(H) domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, C2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

[0212] The antibodies of the present invention may be monospecific,bispecific, trispecific or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of thepresent invention as well as for a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTpublications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt etal., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

[0213] Antibodies of the present invention may be described or specifiedin terms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

[0214] Antibodies of the present invention may also be described orspecified in terms of their cross-reactivity. Antibodies that do notbind any other analog, ortholog, or homolog of a polypeptide of thepresent invention are included. Antibodies that bind polypeptides withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Antibodies that do not bind polypeptides withless than 95%, less than 90%, less than 85%, less than 80%, less than75%, less than 70%, less than 65%, less than 60%, less than 55%, andless than 50% identity (as calculated using methods known in the art anddescribed herein) to a polypeptide of the present invention are alsoincluded in the present invention. Further included in the presentinvention are antibodies that bind polypeptides encoded bypolynucleotides which hybridize to a polynucleotide of the presentinvention under stringent hybridization conditions (as describedherein). Antibodies of the present invention may also be described orspecified in terms of their binding affinity to a polypeptide of theinvention. Preferred binding affinities include those with adissociation constant or Kd less than 5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M,5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M,10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M,10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, and 10⁻¹⁵M.

[0215] The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0216] Antibodies of the present invention may act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. The invention features both receptor-specificantibodies and ligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

[0217] The invention also features receptor-specific antibodies whichboth prevent ligand binding and receptor activation as well asantibodies that recognize the receptor-ligand complex, and, preferably,do not specifically recognize the unbound receptor or the unboundligand. Likewise, included in the invention are neutralizing antibodieswhich bind the ligand and prevent binding of the ligand to the receptor,as well as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. Thus, the inventionfurther relates to antibodies which act as agonists or antagonists ofthe polypeptides of the present invention. The above antibody agonistscan be made using methods known in the art. See, e.g., PCT publicationWO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161:1786-1794 (1998); Zhu etal., Cancer Res. 58:3209-3214(1998); Yoon et al., J. Immunol. 160:3170-3179 (1998); Prat et al., J.Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods205:177-190 (1997); Liautard et al., Cytokine 9:233-241 (1997); Carlsonet al., J. Biol. Chem. 272:11295-11301 (1997); Taryman et al., Neuron14:755-762 (1995); Muller et al., Structure 6:1153-1167 (1998); Bartuneket al., Cytokine 8:14-20 (1996) (which are all incorporated by referenceherein in their entireties).

[0218] Antibodies of the present invention may be used, for example, butnot limited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

[0219] As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

[0220] The antibodies of the invention include derivatives that aremodified, i.e, by the covalent attachment of any type of molecule to theantibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

[0221] The antibodies of the present invention may be generated by anysuitable method known in the art. Polyclonal antibodies to an antigen ofinterest can be produced by various procedures well known in the art.For example, a polypeptide of the invention can be administered tovarious host animals including, but not limited to, rabbits, mice, rats,etc. to induce the production of sera containing polyclonal antibodiesspecific for the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Such adjuvants are also well known in the art.

[0222] Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” is not limited to antibodiesproduced through hybridoma technology. Monoclonal antibodies can beprepared using a wide variety of techniques known in the art includingthe use of hybridoma and recombinant and phage display technology.

[0223] Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 8. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

[0224] Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

[0225] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, Fab and F(ab′)2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

[0226] For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

[0227] As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12:864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0228] Examples of techniques which can be used to produce single-chainFvs and antibodies include those described in U.S. Pat. Nos. 4,946,778and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991);Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science240:1038-1040 (1988). For some uses, including in vivo use of antibodiesin humans and in vitro detection assays, it may be preferable to usechimeric, humanized, or human antibodies. A chimeric antibody is amolecule in which different portions of the antibody are derived fromdifferent animal species, such as antibodies having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region. Methods for producing chimeric antibodies are known inthe art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al.,BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, whichare incorporated herein by reference in their entireties. Humanizedantibodies are antibody molecules from non-human species antibody thatbinds the desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7:805-814 (1994); Roguska.et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No.5,565,332).

[0229] Completely human antibodies are particularly desirable fortherapeutic treatment of human patients. Human antibodies can be made bya variety of methods known in the art including phage display methodsdescribed above using antibody libraries derived from humanimmunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and4,716,111 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893,WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which isincorporated herein by reference in its entirety.

[0230] Human antibodies can also be produced using transgenic mice whichare incapable of expressing functional endogenous immunoglobulins, butwhich can express human immunoglobulin genes. For example, the humanheavy and light chain immunoglobulin gene complexes may be introducedrandomly or by homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

[0231] Completely human antibodies which recognize a selected epitopecan be generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

[0232] Further, antibodies to the polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7:437-444(1989) and Nissinoff, J. Immunol. 147:2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

A. Polynucleotides Encoding Antibodies

[0233] The invention further provides polynucleotides comprising anucleotide sequence encoding an antibody of the invention and fragmentsthereof. The invention also encompasses polynucleotides that hybridizeunder stringent or lower stringency hybridization conditions, e.g., asdefined supra, to polynucleotides that encode an antibody, preferably,that specifically binds to a polypeptide of the invention, preferably,an antibody that binds to a polypeptide having the amino acid sequenceof SEQ ID NO:2 or 4.

[0234] The polynucleotides may be obtained, and the nucleotide sequenceof the polynucleotides determined, by any method known in the art. Forexample, if the nucleotide sequence of the antibody is known, apolynucleotide encoding the antibody may be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,BioTechniques 17:242 (1994)), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligation of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

[0235] Alternatively, a polynucleotide encoding an antibody may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the immunoglobulin may be obtained from a suitable source(e.g., an antibody cDNA library, or a cDNA library generated from, ornucleic acid, preferably poly A+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody of the invention) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

[0236] Once the nucleotide sequence and corresponding amino acidsequence of the antibody is determined, the nucleotide sequence of theantibody may be manipulated using methods well known in the art for themanipulation of nucleotide sequences, e.g., recombinant DNA techniques,site directed mutagenesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, New York, which are both incorporated by referenceherein in their entireties), to generate antibodies having a differentamino acid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

[0237] In a specific embodiment, the amino acid sequence of the heavyand/or light chain variable domains may be inspected to identify thesequences of the complementarity determining regions (CDRs) by methodsthat are well know in the art, e.g., by comparison to known amino acidsequences of other heavy and light chain variable regions to determinethe regions of sequence hypervariability. Using routine recombinant DNAtechniques, one or more of the CDRs may be inserted within frameworkregions, e.g., into human framework regions to humanize a non-humanantibody, as described supra. The framework regions may be naturallyoccurring or consensus framework regions, and preferably human frameworkregions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) fora listing of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

[0238] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855(1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

[0239] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-42(1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);and Ward et al., Nature 334:544-554 (1989)) can be adapted to producesingle chain antibodies. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

B. Methods of Producing Antibodies

[0240] The antibodies of the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or preferably, by recombinant expression techniques.

[0241] Recombinant expression of an antibody of the invention, orfragment, derivative or analog thereof, e.g., a heavy or light chain ofan antibody of the invention, requires construction of an expressionvector containing a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods, which are well known to those skilled in the art, canbe used to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

[0242] The expression vector is transferred to a host cell byconventional techniques and the transfected cells are then cultured byconventional techniques to produce an antibody of the invention. Thus,the invention includes host cells containing a polynucleotide encodingan antibody of the invention, or a heavy or light chain thereof,operably linked to a heterologous promoter. In preferred embodiments forthe expression of double-chained antibodies, vectors encoding both theheavy and light chains may be co-expressed in the host cell forexpression of the entire immunoglobulin molecule, as detailed below.

[0243] A variety of host-expression vector systems maybe utilized toexpress the antibody molecules of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest maybe produced and subsequently purified, but also represent cells whichmay, when transformed or transfected with the appropriate nucleotidecoding sequences, express an antibody molecule of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing antibodycoding sequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

[0244] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

[0245] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

[0246] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the antibody coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non- essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

[0247] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

[0248] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the antibody molecule may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

[0249] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler et al.,Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalski, Bioessays 14:495-500 (1992), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes, whichcan be employed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:3567 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981); neo, which confers resistance to the aminoglycoside G-418(Southern, P. J., et al., J. Mol. Appl. Genet. 1:327-341 (1982)); andhygro, which confers resistance to hygromycin (Santerre et al., Gene30:147 (1984)). Methods commonly known in the art of recombinant DNAtechnology which can be used are described in Ausubel et al. (eds.),1993, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York; and in Chapters 12 and 13, Dracopoli et al.(eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NewYork.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which areincorporated by reference herein in their entireties.

[0250] The expression levels of an antibody molecule can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

[0251] The host cell may be co-transfected with two expression vectorsof the invention, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes both heavy andlight chain polypeptides. In such situations, the light chain should beplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.Sci. USA 77:2197). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

[0252] Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

C. Antibody Conjugates

[0253] The present invention encompasses antibodies recombinantly fusedor chemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

[0254] The present invention further includes compositions comprisingthe polypeptides of the present invention fused or conjugated toantibody domains other than the variable regions. For example, thepolypeptides of the present invention may be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention may comprise the constant region,hinge region, CH1 domain, CH2 domain, and CH3 domain or any combinationof whole domains or portions thereof. The polypeptides may also be fusedor conjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

[0255] As discussed, supra, the polypeptides of the present inventionmay be fused or conjugated to the above antibody portions to increasethe in vivo half-life of the polypeptides or for use in immunoassaysusing methods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinctic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5 receptor, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)).

[0256] Moreover, the antibodies or fragments thereof of the presentinvention can be fused to marker sequences, such as a peptide tofacilitate their purification. In preferred embodiments, the markeramino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

[0257] The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment and/or prevention regimens.Detection can be facilitated by coupling the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 formetal ions, which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidintbiotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

[0258] Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0259] The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0260] Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

[0261] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp.303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

[0262] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980, which is incorporated herein by reference in itsentirety.

[0263] An antibody, with or without a therapeutic moiety conjugated toit, administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

D. Assays For Antibody Binding

[0264] The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

[0265] Immunoprecipitation protocols generally comprise lysing apopulation of cells in a lysis buffer such as RIPA buffer (1% NP-40 orTriton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 Msodium phosphate at pH 7.2, 1% Trayslol) supplemented with proteinphosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin,sodium vanadate), adding the antibody of interest to the cell lysate,incubating for a period of time (e.g., 1-4 hours) at 4° C., addingprotein A and/or protein G sepharose beads to the cell lysate,incubating for about an hour or more at 4° C., washing the beads inlysis buffer and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, e.g., western blot analysis. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the binding of the antibody to an antigen and decrease thebackground (e.g., pre-clearing the cell lysate with sepharose beads).For further discussion regarding immunoprecipitation protocols see,e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0266] Western blot analysis generally comprises preparing proteinsamples, electrophoresis of the protein samples in a polyacrylamide gel(e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), transferring the protein sample from the polyacrylamide gel toa membrane such as nitrocellulose, PVDF or nylon, blocking the membranein blocking solution (e.g., PBS with 3% BSA or non-fat milk), washingthe membrane in washing buffer (e.g., PBS-Tween 20), blocking themembrane with primary antibody (the antibody of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise. For further discussion regarding westernblot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocolsin Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at10.8.1.

[0267] ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

[0268] The binding affinity of an antibody to an antigen and theoff-rate of an antibody-antigen interaction can be determined bycompetitive binding assays. One example of a competitive binding assayis a radioimmunoassay comprising the incubation of labeled antigen(e.g., ³H or ¹²⁵I) with the antibody of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody ofinterest for a particular antigen and the binding off-rates can bedetermined from the data by scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the antigen is incubated with antibody of interest is conjugatedto a labeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody.

E. Antibody Based Therapies

[0269] The present invention is further directed to antibody-basedtherapies which involve administering antibodies of the invention to ananimal, preferably a mammal, and most preferably a human, patient fortreating and/or preventing one or more of the disorders or conditionsdescribed herein. Therapeutic compounds of the invention include, butare not limited to, antibodies of the invention (including fragments,analogs and derivatives thereof as described herein) and nucleic acidsencoding antibodies of the invention (including fragments, analogs andderivatives thereof as described herein).

[0270] While not intending to be bound to theory, DR3 receptors arebelieved to induce programmed cell death by theassociation/cross-linking of death domains between different receptormolecules. Thus, agents (e.g., antibodies) that preventassociation/cross-linking of DR3 death domains will prevent DR3 mediatedprogrammed cell death, and agents (e.g., antibodies) that induceassociation/cross-linking of DR3 death domains will induce DR3 mediatedprogrammed cell death. Further, DR3 ligands (e.g., TNF-γ-β) that induceDR3 mediated programmed cell death are believed to function by causingthe association/cross-linking of DR3 death domains.

[0271] As suggested above, DR3 receptors have been shown to bind TNF-γ-β(see PCT Publication No. WO 00/08139), the entire disclosure of which isincorporated herein by reference). DR3 receptors are also known to bepresent in a number of tissues and on the surfaces of a number of celltypes. These tissues and cell types include endothelial cells, livercells, hepatocellular tumor, lymph nodes, Hodgkin's lymphoma, tonsil,bone marrow, spleen, heart, thymus, pericardium, healing wound (skin),brain, pancreas tumor, burned skin, U937 cells, testis, colon cancer(metastasized to liver), pancreas, rejected kidney, adipose, ovary,olfactory epithelium, striatum depression, HeLa cells, LNCAP (upontreatment with +30 nM androgen), HUVEC (human umbilical vein endothelialcells), 8 week embryo tissues, 9 week embryo tissues, fetal braintissues, fetal kidney tissues, fetal heart tissues, fetal thymustissues, fetal lung tissues, fetal liver tissues, fetal spleen tissues,T-cell helper II, activated T-cell (16 hr), activated T-cell (24 hr),primary dendritic cells, eosinophils, monocytes, and keratinocytes.Further, TNF-γ-β has been shown to induce apoptosis, to haveanti-angiogenic activity, and to inhibit the growth of tumor cells invivo. Additionally, TNF-γ-β activities are believed to be modulated, atleast in part, through interaction with DR3 receptors.

[0272] Antibodies which act as both agonists and antagonists of receptorfunctions are known in the art. For example, Deng et al., (Blood92:1981-1988 (1998)) describe a monoclonal antibody which binds to thehuman c-Mpl receptor and stimulates megakaryocytopoiesis. The monoclonalantibody described in Deng et al. is thus a c-Mpl receptor agonist.

[0273] Antibodies which bind to DR3 receptors will have varying effectson these receptors. These effects differ based on the specific portionsof the DR3 receptor to which the antibodies bind and thethree-dimensional conformation of the antibody molecules themselves.Thus, antibodies which bind to the extracellular domain of a DR3receptor can either stimulate or inhibit DR3 activities (e.g., theinduction of apoptosis). Antibodies which stimulate DR3 receptoractivities (e.g., by facilitating the association between DR3 receptordeath domains) are DR3 agonists and antibodies which inhibit DR3receptor activities (e.g., by blocking the binding of TNF-γ-β and/orpreventing the association between DR3 receptor death domains) are DR3antagonists.

[0274] Antibodies of the invention which function as agonists andantagonists of DR3 receptors include, antigen-binding antibody fragmentssuch as Fab and F(ab′)₂ fragments, Fd, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain, as well as polyclonal, monoclonal and humanizedantibodies. Each of these antigen-binding antibody fragments andantibodies are described in more detail elsewhere herein.

[0275] In view of the above, antibodies of the invention, as well asother agonists, are useful for stimulating DR3 death domain activity inendothelial cells, resulting in anti-angiogenic activity. Antibodies ofthis type are useful for prevention and/or treating diseases andconditions associated with hypervascularization and neovascularization,such as rheumatoid arthritis and solid tissue cancers (e.g., skincancer, head and neck tumors, breast tumors, endothelioma,osteoblastoma, osteoclastoma, and Kaposi's sarcoma), as well as diseasesand conditions associated with chronic inflammation.

[0276] Diseases and conditions associated with chronic inflammation,such as ulcerative colitis and Crohn's disease, often show histologicalchanges associated with the ingrowth of new blood vessels into theinflamed tissues. Agonists of the invention which stimulate the activityof DR3 death domains will induce apoptosis in endothelial cells. As aresult, agonists of the invention can inhibit the formation of blood andlymph vessels and, thus, can be used to prevent and/or treat diseasesand conditions associated with hypervascularization andneovascularization.

[0277] Other diseases and conditions associated with angiogenesis whichcan be prevented and/or treated using agonists of the invention includehypertrophic and keloid scarring, proliferative diabetic retinopathy,arteriovenous malformations, atherosclerotic plaques, hemophilic joints,nonunion fractures, Osler-Weber syndrome, psoriasis, pyogenic granuloma,scleroderma, tracoma, menorrhagia, and vascular adhesions.

[0278] As noted above, DR3 receptors are also found on T-cells. Thus,agonists of the invention (e.g., anti-DR3 receptor antibodies) are alsouseful for inhibiting T-cell mediated immune responses, as well aspreventing and/or treating diseases and conditions associated withincreased T-cell proliferation. Diseases and conditions associated withT-cell mediated immune responses and increased T-cell proliferationinclude graft-v-host responses and diseases, inflammation, autoimmunediseases, and T-cell leukemias.

[0279] Further, agents which inhibit DR3 death domain activity (e.g.,DR3 antagonists) are also useful for preventing and/or treating a numberof diseases and conditions associated with decreased vascularization,decreased T-cell proliferation, and decreases in T-cell populations. Asindicated above, examples of antagonists of DR3 receptor activityinclude anti-DR3 receptor antibodies. These antibodies can function, forexamples, by either binding to DR3 receptors and blocking the binding ofligands which stimulate DR3 death domain activity (e.g., TNF-γ-β) orinhibiting DR3 receptor conformational changes associated with membranesignal transduction.

[0280] An example of a condition associated with decreasedvascularization that can be treated using antagonists of the inventionis delayed wound healing. The elderly, in particular, often heal at aslower rate than younger individuals. Antagonists of the invention canthus prevent and/or inhibit apoptosis from occurring in endothelialcells at wound sites and thereby promote wound healing in healingimpaired individuals, as well as in individuals who heal at “normal”rates. Thus, antagonists of the invention can be used to promote and/oraccelerate wound healing. Antagonists of the invention are also usefulfor treating and/or preventing other diseases and conditions includingrestenosis, myocardial infarction, peripheral arterial disease, criticallimb ischemia, angina, atherosclerosis, ischemia, edema, livercirrhosis, osteoarthritis, and pulmonary fibrosis.

[0281] Antagonists of the invention (e.g., anti-DR3 receptor antibodies)are also useful for enhancing T-cell mediated immune responses, as wellas preventing and/or treating diseases and conditions associated withdecreased T-cell proliferation. Antibodies of the invention which blockthe binding of DR3 receptor ligands to DR3 receptors or interfere withDR3 receptor conformational changes associated with membrane signaltransduction can inhibit DR3 mediated T-cell apoptosis. The inhibitionof DR3 mediated apoptosis can, for examples, either result in anincrease in the expansion rate of in vivo T-cell populations or preventa decrease in the size of such populations. Thus, antagonists of theinvention can be used to prevent and/or treat diseases or conditionsassociated with decreased or decreases in T-cell populations. Examplesof such diseases and conditions included acquired immune deficiencysyndrome (AIDS) and related afflictions (e.g., AIDS related complexes),T-cell immunodeficiencies, radiation sickness, and T-cell depletion dueto radiation and/or chemotherapy.

[0282] Further, when an antagonist of the invention is administered toan individual for the treatment and/or prevention of a disease orcondition associated with decreased T-cell populations, the antagonistmay be co-administered with an agent that activates and/or induceslymphocyte proliferation (e.g., a cytokine). Combination therapies ofthis nature, as well as other combination therapies, are discussed belowin more detail.

[0283] Anti-DR3 antibodies are thus useful for treating and/orpreventing malignancies, abnormalities, diseases and/or conditionsinvolving tissues and cell types which express DR3 receptors. Further,malignancies, abnormalities, diseases and/or conditions which can betreated and/or prevented by the induction of programmed cell death incells which express DR3 receptors can be treated and/or prevented usingDR3 receptor agonists of the invention. Similarly, malignancies,abnormalities, diseases and/or conditions which can be treated and/orprevented by inhibiting programmed cell death in cells which express DR3receptors can be treated and/or prevented using DR3 receptor antagonistsof the invention.

[0284] A number of additional malignancies, abnormalities, diseasesand/or conditions which can be treated using the agonists andantagonists of the invention are set out elsewhere herein, for example,in the section below entitled “Therapeutics”.

[0285] The antibodies of the present invention may be usedtherapeutically in a number of ways, includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g., as mediated bycomplement (CDC) or by effector cells (ADCC).

[0286] The antibodies of this invention may be advantageously utilizedin combination with other monoclonal or chimeric antibodies, or withlymphokines, tumor necrosis factors (e.g., TNF-γ-β) or hematopoieticgrowth factors (e.g., IL-2, IL-3 and IL-7). For example, agonisticanti-DR3 antibodies may be administered in conjunction with TNF-γ-β whenone seeks to induce DR3 mediated cell death in cells that express DR3receptors of the invention. Combination therapies of this nature, aswell as other combination therapies, are discussed below in more detail.

[0287] The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

[0288] It is preferred to use high affinity and/or potent in vivoinhibiting and/or neutralizing antibodies against polypeptides orpolynucleotides of the present invention, fragments or regions thereof,for both immunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

[0289] Transgenic Non-Human Animals

[0290] The proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

[0291] Any technique known in the art may be used to introduce thetransgene (i.e., nucleic acids of the invention) into animals to producethe founder lines of transgenic animals. Such techniques include, butare not limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pluripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each of the documentsrecited in this paragraph are herein incorporated by reference in theirentirety. See also, U.S. Pat. No. 5,464,764 (Capecchi et al.,Positive-Negative Selection Methods and Vectors); U.S. Pat. No.5,631,153 (Capecchi et al., Cells and Non-Human Organisms ContainingPredetermined Genomic Modifications and Positive-Negative SelectionMethods and Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder etal., Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagneret al., Genetic Transformation of Zygotes); each of which is herebyincorporated by reference in its entirety.

[0292] Any technique known in the art may be used to produce transgenicclones containing polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campbell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

[0293] The present invention provides for transgenic animals that carrythe transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the polynucleotide transgene be integratedinto the chromosomal site of the endogenous gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph are hereinincorporated by reference in their entirety.

[0294] Once transgenic animals have been generated, the expression ofthe recombinant gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

[0295] Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

[0296] Transgenic and “knock-out” animals of the invention have useswhich include, but are not limited to, animal model systems useful inelaborating the biological function of DR3-V1 or DR3 polypeptides,studying conditions and/or disorders associated with aberrant DR3-V1 orDR3 expression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

[0297] In further embodiments of the invention, cells that aregenetically engineered to express the proteins of the invention, oralternatively, that are genetically engineered not to express theproteins of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Thecells are genetically engineered in vitro using recombinant DNAtechniques to introduce the coding sequence of polypeptides of theinvention into the cells, or alternatively, to disrupt the codingsequence and/or endogenous regulatory sequence associated with thepolypeptides of the invention, e.g., by transduction (using viralvectors, and preferably vectors that integrate the transgene into thecell genome) or transfection procedures, including, but not limited to,the use of plasmids, cosmids, YACs, naked DNA, electroporation,liposomes, etc. The coding sequence of the polypeptides of the inventioncan be placed under the control of a strong constitutive or induciblepromoter or promoter/enhancer to achieve expression, and preferablysecretion, of the polypeptides of the invention. The engineered cellsthat express and preferably secrete the polypeptides of the inventioncan be introduced into the patient systemically, e.g., in thecirculation, or intraperitoneally. Alternatively, the cells can beincorporated into a matrix and implanted in the body, e.g., geneticallyengineered fibroblasts can be implanted as part of a skin graft;genetically engineered endothelial cells can be implanted as part of alymphatic or vascular graft. (See, e.g., Anderson et al., U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

[0298] When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well-known techniques,which prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form, which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

[0299] Therapeutics

[0300] The Tumor Necrosis Factor (TNF) family ligands are known to beamong the most pleiotropic cytokines, inducing a large number ofcellular responses, including cytotoxicity, anti-viral activity,immunoregulatory activities, and the transcriptional regulation ofseveral genes (D. V. Goeddel et al., “Tumor Necrosis Factors: GeneStructure and Biological Activities,” Symp. Quant. Biol. 51:597-609(1986), Cold Spring Harbor; B. Beutler and A. Cerami, Annu. Rev.Biochem. 57:505-518 (1988); L. J. Old, Sci. Am. 258:59-75 (1988); W.Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands inducesuch various cellular responses by binding to TNF-family receptors,including the DR3-V1 or DR3 of the present invention.

[0301] Cells which express the DR3-V1 or DR3 polypeptide and arebelieved to have a potent cellular response to DR3-V1 or DR3 ligandsinclude lymphocytes, fibroblasts, macrophages, synovial cells, activatedT-cells, lymphoblasts and epithelial cells. By “a cellular response to aTNF-family ligand” is intended any genotypic, phenotypic, and/ormorphologic change to a cell, cell line, tissue, tissue culture orpatient that is induced by a TNF-family ligand. As indicated, suchcellular responses include not only normal physiological responses toTNF-family ligands, but also diseases associated with increasedapoptosis or the inhibition of apoptosis. Apoptosis—programmed celldeath—is a physiological mechanism involved in the deletion ofperipheral T lymphocytes of the immune system, and its dysregulation canlead to a number of different pathogenic processes (J. C. Ameisen, AIDS8:1197-1213 (1994); P. H. Krammer et al., Curr. Opin. Immunol. 6:279-289(1994)).

[0302] DR3-V1 or DR3 polynucleotides, polypeptides, agonists orantagonists of the invention may be used in developing treatments anddiagnostic/prognostic assays for any disorder mediated (directly orindirectly) by defective, or insufficient amounts of DR3. DR3-V1 or DR3polypeptides, agonists or antagonists may be administered to a patient(e.g., mammal, preferably human) afflicted with such a disorder.Alternatively, a gene therapy approach may be applied to treat and/orprevent such disorders. Disclosure herein of DR3-V1 or DR3 nucleotidesequences permits the detection of defective DR3 genes, and thereplacement thereof with normal DR3-encoding genes. Defective genes maybe detected in in vitro diagnostic assays, and by comparison of theDR3-V1 or DR3 nucleotide sequence disclosed herein with that of a DR3gene derived from a patient suspected of harboring a defect in thisgene.

[0303] Diseases associated with increased cell survival, or theinhibition of apoptosis, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, such asbreast cancer, prostrate cancer, Kaposi sarcoma and ovarian cancer);autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus, immune-relatedglomerulonephritis, and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), information graftversus host disease, acute graft rejection, and chronic graft rejection.Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration); myelodysplastic syndromes (such as aplastic anemia),ischemic injury (such as that caused by myocardial infarction, strokeand reperfusion injury), toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia, and anorexia.

[0304] Additional diseases or conditions associated with increased cellsurvival include, but are not limited to, progression, and/or metastasesof malignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

[0305] Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration and brain tumor or prior associated disease); autoimmunedisorders (such as, multiple sclerosis, Sjogren's syndrome, Grave'sdisease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,Behcet's disease, Crohn's disease, polymyositis, systemic lupuserythematosus, immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, and rheumatoid arthritis) myelodysplasticsyndromes (such as aplastic anemia), graft vs. host disease (acuteand/or chronic), ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury or disease(e.g., hepatitis related liver injury, cirrhosis, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, ulcerativecolitis, cachexia and anorexia. In preferred embodiments, DR3polynucleotides, polypeptides, agonists, and/or antagonists are used totreat, prevent, diagnose and/or prognose the diseases and disorderslisted above.

[0306] Thus, in one aspect, the present invention is directed to amethod for enhancing apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell which expresses the DR3-V1 or DR3polypeptide an effective amount of DR3-V1 or DR3 ligand, analog or anagonist capable of increasing DR3-V1 or DR3 mediated signaling.Preferably, DR3-V1 or DR3 mediated signaling is increased to treatand/or prevent a disease wherein decreased apoptosis or decreasedcytokine and adhesion molecule expression is exhibited. An agonist caninclude soluble forms of DR3-V1 or DR3 and monoclonal antibodiesdirected against the DR3-V1 or DR3 polypeptide.

[0307] In a further aspect, the present invention is directed to amethod for inhibiting apoptosis induced by a TNF-family ligand, whichinvolves administering to a cell that expresses the DR3-V1 or DR3polypeptide an effective amount of an antagonist capable of decreasingDR3-V1 or DR3 mediated signaling. Preferably, DR3-V1 or DR3 mediatedsignaling is decreased to treat and/or prevent a disease whereinincreased apoptosis or NF-kB expression is exhibited. An antagonist caninclude soluble forms of DR3-V1 or DR3 and monoclonal antibodiesdirected against the DR3-V1 or DR3 polypeptide.

[0308] In one more particular aspect, the present invention is directedto compositions and methods useful for treating, preventing and/ordiagnosing diseases wherein decreased apoptosis of T-cells is exhibited.Examples of such diseases include, but are not limited to, graft vs.host disease (acute and/or chronic), multiple sclerosis, Sjogren'ssyndrome, Grave's disease, Hashimoto's thyroiditis, autoimmune diabetes,biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,systemic lupus erythematosus, immune-related glomerulonephritis,autoimmune gastritis, thrombocytopenic purpura, rheumatoid arthritis andulcerative colitis.

[0309] In a further particular aspect, the present invention is directedto compositions and methods useful for treating, preventing and/ordiagnosing diseases wherein increased secretion of proinflammatorycytokines (e.g., IFN-γ) is exhibited. Examples of such diseases include,but are not limited to, graft vs. host disease (acute and/or chronic),multiple sclerosis, Sjogren's syndrome, Grave's disease, Hashimoto'sthyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease,Crohn's disease, polymyositis, systemic lupus erythematosus,immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, rheumatoid arthritis and ulcerative colitis.

[0310] In another aspect, DR3-V1-Fc and DR3-Fc proteins and solubleportions of the extracellular domains of DR3-V1 and DR3 proteins areuseful in stimulating neovascularization and angiogenesis. Thus, thesepolypeptides are useful, for example, for the treatment and/orprevention of diseases and conditions associated withhypovascularization (e.g., Turner's syndrome, cardiovascular aging,bronchial stenosis, depression).

[0311] Specifically included within the scope of the invention areDR3-V1-Fc and DR3-Fc proteins receptor/Fc fusion proteins, and nucleicacid molecules that encode such proteins. These fusion proteins includethose having amino acid sequences of the extracellular domains of theDR3 proteins of the invention. Examples of portions of DR3 extracellulardomains which are useful in the preparation of DR3 receptor/Fc fusionproteins include amino acids 1 to 199 in SEQ ID NO:4 and amino acids 1to 210, 37 to 210, 50 to 210, and 100 to 210 in SEQ ID NO:2.

[0312] In one more particular aspect, DR3-V1-Fc and DR3-Fc proteins andsoluble portions of the extracellular domains of DR3-V1 and DR3 proteinsare useful for treating, preventing and/or diagnosing diseases whereindecreased apoptosis of T-cells is exhibited. Examples of such diseasesinclude, but are not limited to, graft vs. host disease (acute and/orchronic), multiple sclerosis, Sjogren's syndrome, Grave's disease,Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis,Behcet's disease, Crohn's disease, polymyositis, systemic lupuserythematosus, immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, rheumatoid arthritis and ulcerative colitis.

[0313] In another aspect, DR3-V1-Fc and DR3-Fc proteins and solubleportions of the extracellular domains of DR3-V 1 and DR3 proteins areuseful for treating, preventing and/or diagnosing diseases whereinincreased secretion of proinflammatory cytokines (e.g., IFN-γ) isexhibited. Examples of such diseases include, but are not limited to,graft vs. host disease (acute and/or chronic), multiple sclerosis,Sjogren's syndrome, Grave's disease, Hashimoto's thyroiditis, autoimmunediabetes, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus, immune-relatedglomerulonephritis, autoimmune gastritis, thrombocytopenic purpura,rheumatoid arthritis and ulcerative colitis.

[0314] Further, afflictions which can be treated and/or prevented byDR3-V1 and DR3 mediated stimulation of angiogenesis include soft tissuetraumas (e.g., cuts and bruises), ulcers (e.g., peptic, skin andvenous), and sclerodermas.

[0315] By “agonist” is intended, naturally occurring and syntheticcompounds capable of enhancing or potentiating apoptosis. By“antagonist” is intended, naturally occurring and synthetic compoundscapable of inhibiting apoptosis. Whether any candidate “agonist” or“antagonist” of the present invention can enhance or inhibit apoptosiscan be determined using art-known TNF-family ligand/receptor cellularresponse assays, including those described in more detail below.

[0316] One such screening procedure involves the use of melanophores,which are transfected to express the receptor of the present invention.Such a screening technique is described in PCT WO 92/01810, publishedFeb. 6, 1992. Such an assay may be employed, for example, for screeningfor a compound that inhibits (or enhances) activation of the receptorpolypeptide of the present invention by contacting the melanophorecells, which encode the receptor with both a TNF-family ligand and thecandidate antagonist (or agonist). Inhibition or enhancement of thesignal generated by the ligand indicates that the compound is anantagonist or agonist of the ligand/receptor signaling pathway.

[0317] Other screening techniques include the use of cells which expressthe receptor (for example, transfected CHO cells) in a system whichmeasures extracellular pH changes caused by receptor activation, forexample, as described in Science 246:181-296 (October 1989). Forexample, compounds may be contacted with a cell which expresses thereceptor polypeptide of the present invention and a second messengerresponse, e.g., signal transduction or pH changes, may be measured todetermine whether the potential compound activates or inhibits thereceptor.

[0318] Another such screening technique involves introducing RNAencoding the receptor into Xenopus oocytes to transiently express thereceptor. The receptor oocytes may then be contacted with the receptorligand and a compound to be screened, followed by detection ofinhibition or activation of a calcium signal in the case of screeningfor compounds that are thought to inhibit activation of the receptor.

[0319] Another screening technique involves expressing in cells aconstruct wherein the receptor is linked to a phospholipase C or D. Suchcells include endothelial cells, smooth muscle cells, embryonic kidneycells, etc. The screening may be accomplished as herein above describedby detecting activation of the receptor or inhibition of activation ofthe receptor from the phospholipase signal.

[0320] Another method involves screening for compounds that inhibitactivation of the receptor polypeptide of the present inventionantagonists by determining inhibition of binding of labeled ligand tocells that have the receptor on the surface thereof. Such a methodinvolves transfecting a eukaryotic cell with DNA encoding the receptorsuch that the cell expresses the receptor on its surface and contactingthe cell with a compound in the presence of a labeled form of a knownligand. The ligand can be labeled, e.g., by radioactivity. The amount oflabeled ligand bound to the receptors is measured, e.g., by measuringradioactivity of the receptors. If the compound binds to the receptor,as determined by a reduction of labeled ligand that binds to thereceptors, the binding of labeled ligand to the receptor is inhibited.

[0321] Further screening assays for agonist and antagonist of thepresent invention are described in L. A. Tartaglia and D. V. Goeddel, J.Biol. Chem. 267:4304-4307(1992).

[0322] Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express the DR3-V1 or DR3polypeptide with a candidate compound and a TNF-family ligand, assayinga cellular response, and comparing the cellular response to a standardcellular response, the standard being assayed when contact is made withthe ligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing the DR3-V1 or DR3 polypeptide can becontacted with either an endogenous or exogenously administeredTNF-family ligand.

[0323] Agonist according to the present invention include naturallyoccurring and synthetic compounds such as, for example, TNF familyligand peptide fragments, transforming growth factor β,neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate),tumor suppressors (p53), cytolytic T cells and antimetabolites.Preferred agonist include chemotherapeutic drugs such as, for example,cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogenmustard, methotrexate and vincristine. Others include ethanol andβ-amyloid peptide (Science 267:1457-1458 (1995)). Further preferredagonists include polyclonal and monoclonal antibodies raised against theDR3-V1 or DR3 polypeptide, or a fragment thereof. Such agonistantibodies raised against a TNF-family receptor are disclosed in L. A.Tartaglia et al., Proc. Natl. Acad. Sci. USA 88:9292-9296 (1991); and L.A. Tartaglia and D. V. Goeddel, supra. See, also, PCT Application WO94/09137.

[0324] Antagonists according to the present invention include naturallyoccurring and synthetic compounds such as, for example, the CD40 ligand,neutral amino acids, zinc, estrogen, androgens, viral genes (such asAdenovirus ElB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barrvirus BHRF1, LMP-1, African swine fever virus LMW5-HL, and Herpes virusyl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumorpromoters (such as PMA, Phenobarbital, and α-Hexachlorocyclohexane).

[0325] Other potential antagonists include antisense molecules. Thus, inspecific embodiments, antagonists according to the present invention arenucleic acids corresponding to the sequences contained in DR3-V1 or DR3,or the complementary strand thereof, and/or to nucleotide sequencescontained in the deposited cDNAs having ATCC Deposit No. 97456 and97757. In one embodiment, antisense sequence is generated internally bythe organism, in another embodiment, the antisense sequence isseparately administered (see, for example, O'Connor, J., Neurochem.56:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Antisense technology canbe used to control gene expression through antisense DNA or RNA, orthrough triple-helix formation. Antisense techniques are discussed forexample, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance, Lee etal., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methodsare based on binding of a polynucleotide to a complementary DNA or RNA.

[0326] For example, the 5′ coding portion of a polynucleotide thatencodes the mature polypeptide of the present invention may be used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

[0327] In one embodiment, the DR3-V1 or DR3 antisense nucleic acid ofthe invention is produced intracellularly by transcription from anexogenous sequence. For example, a vector or a portion thereof, istranscribed, producing an antisense nucleic acid (RNA) of the invention.Such a vector would contain a sequence encoding the DR3-V1 or DR3antisense nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others know in the art, used for replication andexpression in vertebrate cells. Expression of the sequence encodingDR3-V1 or DR3, or fragments thereof, can be by any promoter known in theart to act in vertebrate, preferably human cells. Such promoters can beinducible or constitutive. Such promoters include, but are not limitedto, the SV40 early promoter region (Bernoist and Chambon, Nature29:304-310 (1981), the promoter contained in the 3′ long terminal repeatof Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), theherpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441-1445 (1981), the regulatory sequences of the metallothioneingene (Brinster et al., Nature 296:39-42 (1982)), etc.

[0328] The antisense nucleic acids of the invention comprise, oralternatively consist of, a sequence complementary to at least a portionof an RNA transcript of a DR3 gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded DR3-V1 or DR3 antisensenucleic acids, a single strand of the duplex DNA may thus be tested, ortriplex formation may be assayed. The ability to hybridize will dependon both the degree of complementarity and the length of the antisensenucleic acid Generally, the larger the hybridizing nucleic acid, themore base mismatches with a DR3-V1 or DR3 RNA it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

[0329] Oligonucleotides that are complementary to the 5′ end of themessage, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of the DR3-V1 or DR3 shownin SEQ ID NO:2 and SEQ ID NO:4 could be used in an antisense approach toinhibit translation of endogenous DR3-V1 or DR3 mRNA. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention. Whetherdesigned to hybridize to the 5′-, 3′- or coding region of DR3-V1 or DR3mRNA, antisense nucleic acids should be at least six nucleotides inlength, and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects the oligonucleotide is atleast about 10 nucleotides, at least about 17 nucleotides, at leastabout 25 nucleotides or at least about 50 nucleotides. In this context“about” includes the particularly recited value and values larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides.

[0330] The polynucleotides of the invention can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone, for example, toimprove stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci. 84:648-652 (1987); PCT Publication No.WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents (See, e.g., Krol et al.,BioTechniques 6:958-976 (1988)), or intercalating agents (See, e.g.,Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleotide maybe conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

[0331] The antisense oligonucleotide may comprise at least one modifiedbase moiety which is selected from the group including, but not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine,inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,β-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0332] The antisense oligonucleotide may also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0333] In yet another embodiment, the antisense oligonucleotidecomprises at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0334] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)) orachimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

[0335] Polynucleotides of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

[0336] While antisense nucleotides complementary to the DR3-V1 or DR3coding region sequence could be used, those complementary to thetranscribed untranslated region are most preferred.

[0337] Potential antagonists according to the invention also includecatalytic RNA, or a ribozyme (See, e.g., PCT International PublicationWO 90/11364, published Oct. 4, 1990; Sarver et al., Science247:,1222-1225 (1990). While ribozymes that cleave mRNA at site-specificrecognition sequences can be used to destroy DR3-V1 or DR3 mRNAs, theuse of hammerhead ribozymes is preferred. Hammerhead ribozymes cleavemRNAs at locations dictated by flanking regions that form complementarybase pairs with the target mRNA. The sole requirement is that the targetmRNA have the following sequence of two bases: 5′-UG-3′. Theconstruction and production of hammerhead ribozymes is well known in theart and is described more fully in Haseloff and Gerlach, Nature334:585-591 (1988). There are numerous potential hammerhead ribozymecleavage sites within the nucleotide sequence of DR3-V1 (SEQ ID NO:2) orDR3 (SEQ ID NO:4). Preferably, the ribozyme is engineered so that thecleavage recognition site is located near the 5′ end of the DR3-V1 orDR3 mRNA; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

[0338] As in the antisense approach, the ribozymes of the invention canbe composed of modified oligonucleotides (e.g., for improved stability,targeting, etc.) and should be delivered to cells that express DR3-V1 orDR3 in vivo. DNA constructs encoding the ribozyme may be introduced intothe cell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous DR3-V1 or DR3 messages and inhibittranslation. Since ribozymes unlike antisense molecules, are catalytic,a lower intracellular concentration is required for efficiency.

[0339] Endogenous gene expression can also be reduced by inactivating or“knocking out,” the DR3 gene and/or its promoter using targetedhomologous recombination. (See, e.g., Smithies et al., Nature317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompsonet al., Cell 5:313-321 (1989); each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functionalpolynucleotide of the invention (or a completely unrelated DNA sequence)flanked by DNA homologous to the endogenous polynucleotide sequence(either the coding regions or regulatory regions of the gene) can beused, with or without a selectable marker and/or a negative selectablemarker, to transfect cells that express polypeptides of the invention invivo. In another embodiment, techniques known in the art are used togenerate knockouts in cells that contain, but do not express the gene ofinterest. Insertion of the DNA construct, via targeted homologousrecombination, results in inactivation of the targeted gene. Suchapproaches are particularly suited in research and agricultural fieldswhere modifications to embryonic stem cells can be used to generateanimal offspring with an inactive targeted gene (see, e.g., Thomas &Capecchi 1987 and Thompson 1989, supra). However this approach can beroutinely adapted for use in humans provided the recombinant DNAconstructs are directly administered or targeted to the required site invivo using appropriate viral vectors that will be apparent to those ofskill in the art. The contents of each of the documents recited in thisparagraph are herein incorporated by reference in their entireties.

[0340] In other embodiments, antagonists according to the presentinvention include soluble forms of DR3-V1 or DR3 (e.g., fragments of theDR3-V1 shown in SEQ ID NO:2 and DR3 shown in SEQ ID NO:4) that includethe ligand binding domain from the extracellular region of the fulllength receptor. Such soluble forms of the DR3-V1 or DR3, which may benaturally occurring or synthetic, antagonize DR3-V1 or DR3 mediatedsignaling by competing with the cell surface bound forms of the receptorfor binding to TNF-family ligands. Antagonists of the present inventionalso include antibodies specific for TNF-family ligands and bothDR3-V1-Fc and DR3-Fc fusion proteins.

[0341] In one particular aspect, soluble forms of DR3-V1 or DR3(e.g.,fragments of the DR3-V1 shown in SEQ ID NO:2 and DR3 shown in SEQID NO:4) that include the ligand binding domain from the extracellularregion of the full length receptor are useful for treating, preventingand/or diagnosing diseases wherein decreased apoptosis of T-cells isexhibited. Examples of such diseases include, but are not limited to,graft vs. host disease (acute and/or chronic), multiple sclerosis,Sjogren's syndrome, Grave's disease, Hashimoto's thyroiditis, autoimmunediabetes, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus, immune-relatedglomerulonephritis, autoimmune gastritis, thrombocytopenic purpura,rheumatoid arthritis and ulcerative colitis.

[0342] In another aspect, soluble forms of DR3-V1 or DR3 (e.g.,fragments of the DR3-V1 shown in SEQ ID NO:2 and DR3 shown in SEQ IDNO:4) that include the ligand binding domain from the extracellularregion of the full length receptor are useful for treating, preventingand/or diagnosing diseases wherein increased secretion ofproinflammatory cytokines (e.g., IFN-γ) is exhibited. Examples of suchdiseases include, but are not limited to, graft vs. host disease (acuteand/or chronic), multiple sclerosis, Sjogren's syndrome, Grave'sdisease, Hashimoto's thyroiditis, autoimmune diabetes, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus, immune-related glomerulonephritis, autoimmunegastritis, thrombocytopenic purpura, rheumatoid arthritis and ulcerativecolitis.

[0343] By a “TNF-family ligand” is intended naturally occurring,recombinant, and synthetic ligands that are capable of binding to amember of the TNF receptor family and inducing and/or blocking theligand/receptor signaling pathway. Members of the TNF ligand familyinclude, but are not limited to, TNF-α, lymphotoxin-α (LT-α, also knownas TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL, TNF-γ(International Publication No. WO 96/14328), TNF-γ-α, TNF-γ-β(International Publication No. WO 00/08139), AIM-I (InternationalPublication No. WO 97/33899), AIM-II (International Publication No. WO97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-α(International Publication No. WO 98/07880), neutrokine-α (InternationalPublication No. WO 98/18921), CD40L, CD27L, CD30L, 4-1BBL, OX40L andnerve growth factor (NGF).

[0344] Antibodies according to the present invention may be prepared byany of a variety of standard methods using DR3-V1 or DR3 receptorimmunogens of the present invention. Such DR3-V1 or DR3 receptorimmunogens include the DR3-V1 protein shown in SEQ ID NO:2 and the DR3protein shown in SEQ ID NO:4 (each of which may or may not include aleader sequence) and polypeptide fragments of the receptor comprising,or alternatively consisting of, the ligand binding, extracellular,transmembrane, the intracellular domains of DR3-V1 or DR3, or anycombination thereof.

[0345] Polyclonal and monoclonal antibody agonists or antagonistsaccording to the present invention can be raised according to themethods disclosed herein and/or known in the art, such as, for example,those methods described in Tartaglia and Goeddel, J. Biol. Chem.267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216 (1993)), andPCT Application WO 94/09137 (the contents of each of these threeapplications are herein incorporated by reference in their entireties),and are preferably specific to polypeptides of the invention having theamino acid sequence of SEQ ID NOs:2 or 4.

[0346] In one particular aspect, polyclonal and monoclonal antibodyagonists or antagonists according to the present invention are usefulfor treating, preventing and/or diagnosing diseases wherein decreasedapoptosis of T-cells is exhibited. Examples of such diseases include,but are not limited to, graft vs. host disease (acute and/or chronic),multiple sclerosis, Sjogren's syndrome, Grave's disease, Hashimoto'sthyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease,Crohn's disease, polymyositis, systemic lupus erythematosus,immune-related glomerulonephritis, autoimmune gastritis,thrombocytopenic purpura, rheumatoid arthritis and ulcerative colitis.

[0347] In another aspect, polyclonal and monoclonal antibody agonists orantagonists according to the present invention are useful for treating,preventing and/or diagnosing diseases wherein increased secretion ofproinflammatory cytokines (e.g., IFN-γ) is exhibited. Examples of suchdiseases include, but are not limited to, graft vs. host disease (acuteand/or chronic), multiple sclerosis, Sjogren's syndrome, Grave'sdisease, Hashimoto's thyroiditis, autoimmune diabetes, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus, immune-related glomerulonephritis, autoimmunegastritis, thrombocytopenic purpura, rheumatoid arthritis and ulcerativecolitis.

[0348] Further antagonist according to the present invention includesoluble forms of DR3-V1 or DR3, i.e., DR3-V1 or DR3 fragments thatinclude the ligand binding domain from the extracellular region of thefull length receptor. Such soluble forms of the receptor, which may benaturally occurring or synthetic, antagonize DR3-V1 or DR3 mediatedsignaling by competing with the cell surface DR3 -V1 or DR3 for bindingto TNF-family ligands. Thus, soluble forms of the receptor that includethe ligand-binding domain are novel cytokines capable of inhibitingapoptosis induced by TNF-family ligands. These are preferably expressedas dimers or trimers, since these have been shown to be superior tomonomeric forms of soluble receptor as antagonists, e.g., IgG-Fc-TNFreceptor family fusions. Other such cytokines are known in the art andinclude Fas B (a soluble form of the mouse Fas receptor) that actsphysiologically to limit apoptosis induced by Fas ligand (D. P. Hughesand I. N. Crispe, J. Exp. Med. 182:1395-1401 (1995)).

[0349] The experiments set forth in Examples 6 and 7 demonstrate thatDR3 is a death domain-containing molecule capable of triggering bothapoptosis and NF-kB activation, two pathways dominant in the regulationof the immune system. The experiments also demonstrate the internalsignal transduction machinery of this novel cell death receptor. Inaddition, the experiments set forth below demonstrate that DR3-inducedapoptosis was blocked by the inhibitors of ICE-like proteases, CrmA andz-VAD-fmk. Importantly, apoptosis induced by DR3 was also blocked bydominant negative versions of FADD (FADD-DN) or FLICE(FLICE-DN/MACHa1C360S), which were previously shown to inhibit deathsignaling by Fas/APO-1 and TNFR-1. Thus, inhibitors of ICE-likeproteases, FADD-DN and FLICE-DN/MACHa1C360S could also be used asantagonists for DR3 activity.

[0350] The term “antibody” (Ab) or “monoclonal antibody” (mAb) as usedherein is meant to include intact molecules as well as fragments thereof(such as, for example, Fab and F(ab′)₂ fragments) which are capable ofbinding an antigen. Fab and F (ab′)₂ fragments lack the Fc fragment ofintact antibody, clear more rapidly from the circulation, and may haveless non-specific tissue binding of an intact antibody (Wahl et al., J.Nucl. Med. 24:316-325 (1983)).

[0351] Antibodies according to the present invention may be prepared byany of a variety of methods using DR3-V1 or DR3 immunogens of thepresent invention. As indicated, such DR3-V1 or DR3 immunogens includethe full length DR3-V1 or DR3 polypeptide (which may or may not includethe leader sequence) and DR3-V1 or DR3 polypeptide fragments such as theligand-binding domain, the transmembrane domain, the intracellulardomain and the death domain.

[0352] Proteins and other compounds that bind the DR3-V1 or DR3 domainsare also candidate agonist and antagonist according to the presentinvention. Such binding compounds can be “captured” using the yeasttwo-hybrid system (Fields and Song, Nature 340:245-246 (1989)). Amodified version of the yeast two-hybrid system has been described byRoger Brent and his colleagues (J. Gyuris et al., Cell 75:791-803(1993); A. S. Zervos et al., Cell 72:223-232 (1993)). Preferably, theyeast two-hybrid system is used according to the present invention tocapture compounds that bind to either the DR3-V1 or DR3 ligand-bindingdomain or to the DR3-V1 or DR3 intracellular domain. Such compounds aregood candidate agonist and antagonist of the present invention.

[0353] By a “TNF-family ligand” is intended naturally occurring,recombinant, and synthetic ligands that are capable of binding to amember of the TNF receptor family and inducing the ligand/receptorsignaling pathway. Members of the TNF ligand family include, but are notlimited to, the DR3-V1 or DR3 ligand, TNF-α, lymphotoxin-α (LT-α, alsoknown as TNF-β), (International Publication No. WO 96/14328), TNF-γ-α(PCT Publication No. WO 00/08139), TNF-γ-β (PCT Publication No. WO00/08139), LT-β (found in complex heterotrimer LT-α2-β), FasL, CD40,CD27, CD30, 4-1BB, OX40, and nerve growth factor (NGF).

[0354] Representative therapeutic applications of the present inventionare discussed in-more detail below. The state of immunodeficiency thatdefines AIDS is secondary to a decrease in the number and function ofCD4⁺ T-lymphocytes. Recent reports estimate the daily loss of CD4⁺ Tcells to be between 3.5×10⁷ and 2×10⁹ cells (X. Wei et al., Nature373:117-122 (1995)). One cause of CD4⁺ T cell depletion in the settingof HIV infection is believed to be HIV-induced apoptosis. Indeed,HIV-induced apoptotic cell death has been demonstrated not only in vitrobut also, more importantly, in infected individuals (J. C. Ameisen, AIDS8:1197-1213 (1994); T. H. Finkel and N. K. Banda, Curr. Opin. Immunol.6:605-615(1995); C. A. Muro-Cacho et al., J. Immunol. 154:5555-5566(1995)). Furthermore, apoptosis and CD4⁺ T-lymphocyte depletion aretightly correlated in different animal models of AIDS (T. Brunner etal., Nature 373:441-444 (1995); M. L. Gougeon et al., AIDS Res. Hum.Retroviruses 9:553-563 (1993)), and apoptosis is not observed in thoseanimal models in which viral replication does not result in AIDS. Id.Further data indicates that uninfected but primed or activated Tlymphocytes from HIV-infected individuals undergo apoptosis afterencountering the TNF-family ligand FasL. Using monocytic cell lines thatresult in death following HIV infection, it has been demonstrated thatinfection of U937 cells with HIV results in the de novo expression ofFasL and that FasL mediates HIV-induced apoptosis (A. D. Badley et al.,J. Virol. 70:199-206 (1996)). Further, the TNF-family ligand wasdetectable in uninfected macrophages and its expression was upregulatedfollowing HIV infection resulting in selective killing of uninfected CD4T-lymphocytes. Id. Thus, by the invention, a method for treating HIV⁺individuals is provided which involves administering an antagonist ofthe present invention to reduce selective killing of CD4 T-lymphocytes.Modes of administration and dosages are discussed in detail below.

[0355] In rejection of an allograft, the immune system of the recipientanimal has not previously been primed to respond because the immunesystem for the most part is only primed by environmental antigens.Tissues from other members of the same species have not been presentedin the same way that, for example, viruses and bacteria have beenpresented. In the case of allograft rejection, immunosuppressiveregimens are designed to prevent the immune system from reaching theeffector stage. However, the immune profile of xenograft rejection mayresemble disease recurrence more that allograft rejection. In the caseof disease recurrence, the immune system has already been activated, asevidenced by destruction of the native islet cells. Therefore, indisease recurrence the immune system is already at the effector stage.Agonist of the present invention are able to suppress the immuneresponse to both allografts and xenografts because lymphocytes activatedand differentiated into effector cells will express the DR3-V1 or DR3polypeptide, and thereby are susceptible to compounds which enhanceapoptosis. Thus, the present invention further provides a method forcreating immune privileged tissues. Antagonist of the invention canfurther be used in the treatment and/or prevention of InflammatoryBowel-Disease.

[0356] DR3, like TNFR1, also activates the NF-kB transcription factor,which is very closely associated with the stimulation of cytokine (e.g.,IL-8) and adhesion molecule (e.g., ELAM) transcription. Hence, like TNF,the ligand (or agonist) for DR3 and DR3-V1 may in some circumstances beproinflammatory, and antagonists may be useful reagents for blockingthis response. Thus, DR3 and DR3-V1 antagonists may be useful fortreating, preventing, diagnosing and/or prognosing inflammatorydiseases, such as rheumatoid arthritis, osteoarthritis, psoriasis,septicemia, and inflammatory bowel disease.

[0357] In addition, due to lymphoblast expression of DR3, soluble DR3,agonist or antagonist mABs may be used to diagnose, prognose, treatand/or prevent this form of cancer. Further, soluble DR3 or neutralizingmABs may be used to treat and/or prevent various chronic and acute formsof inflammation such as rheumatoid arthritis, osteoarthritis, psoriasis,septicemia, and inflammatory bowel disease.

[0358] DR3 polynucleotides, polypeptides, agonists or antagonists of theinvention may be used to diagnose, prognose, treat and/or preventcardiovascular disorders, including peripheral artery disease, such aslimb ischemia.

[0359] Cardiovascular disorders include cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

[0360] Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

[0361] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

[0362] Heart valve disease include aortic valve insufficiency, aorticvalve stenosis, hear murmurs, aortic valve prolapse, mitral valveprolapse, tricuspid valve prolapse, mitral valve insufficiency, mitralvalve stenosis, pulmonary atresia, pulmonary valve insufficiency,pulmonary valve stenosis, tricuspid atresia, tricuspid valveinsufficiency, and tricuspid valve stenosis.

[0363] Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

[0364] Myocardial ischemias include coronary disease, such as anginapectoris, coronary aneurysm, coronary arteriosclerosis, coronarythrombosis, coronary vasospasm, myocardial infarction and myocardialstunning.

[0365] Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

[0366] Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

[0367] Arterial occlusive diseases include arteriosclerosis,intermittent claudication, carotid stenosis, fibromuscular dysplasias,mesenteric vascular occlusion, Moyamoya disease, renal arteryobstruction, retinal artery occlusion, and thromboangiitis obliterans.

[0368] Cerebrovascular disorders include carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

[0369] Embolisms include air embolisms, amniotic fluid embolisms,cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonaryembolisms, and thromboembolisms. Thrombosis include coronary thrombosis,hepatic vein thrombosis, retinal vein occlusion, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, andthrombophlebitis.

[0370] Ischemia includes cerebral ischemia, ischemic colitis,compartment syndromes, anterior compartment syndrome, myocardialischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitisincludes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

[0371] In one embodiment, a DR3 polynucleotide, polypeptide, agonist, orantagonist of the invention is used to diagnose, prognose, treat and/orprevent thrombotic microangiopathies. One such disorder is thromboticthrombocytopenic purpura (TTP) (Kwaan, H. C., Semin. Hematol. 24:71(1987); Thompson et al., Blood 80:1890 (1992)). IncreasingTTP-associated mortality rates have been reported by the U.S. Centersfor Disease Control (Torok et al., Am. J. Hematol. 50:84 (1995)). Plasmafrom patients afflicted with TTP (including HIV+ and HIV− patients)induces apoptosis of human endothelial cells of dermal microvascularorigin, but not large vessel origin (Laurence et al., Blood 87:3245(1996)). Plasma of TTP patients thus is thought to contain one or morefactors that directly or indirectly induce apoptosis. Another thromboticmicroangiopathy is hemolytic-uremic syndrome (HUS) (Moake, J. L.,Lancet, 343:393,1994; Melnyk et al., (Arch. Intern. Med., 155:2077,1995; Thompson et al., supra). Thus, in one embodiment, the invention isdirected to use of DR3 to diagnose, prognose, treat and/or prevent thecondition that is often referred to as “adult HUS” (even though it canstrike children as well). A disorder known aschildhood/diarrhea-associated HUS differs in etiology from adult HUS. Inanother embodiment conditions characterized by clotting of small bloodvessels may be diagnosed, prognosed, treated and/or prevented using DR3.Such conditions include, but are not limited to, those described herein.For example, cardiac problems seen in about 5-10% of pediatric AIDSpatients are believed to involve clotting of small blood vessels.Breakdown of the microvasculature in the heart has been reported inmultiple sclerosis patients. As a further example, treatment,prevention, diagnosis and/or prognosis of systemic lupus erythematosus(SLE) is contemplated.

[0372] DR3 polynucleotides, polypeptides, agonists or antagonists of theinvention may be employed in combination with other agents useful intreating, preventing, diagnosing and/or prognosing a particulardisorder. For example, in an in vitro study reported by Laurence et al.(Blood 87:3245 (1996)), some reduction of TTP plasma-mediated apoptosisof microvascular endothelial cells was achieved by using an anti-Fasblocking antibody, aurintricarboxylic acid, or normal plasma depleted ofcryoprecipitate. Thus, a patient may be treated with a polynucleotideand/or polypeptide of the invention in combination with an agent thatinhibits Fas-ligand-mediated apoptosis of endothelial cells, such as,for example, an agent described above. In one embodiment, a DR3polynucleotide, polypeptide, agonist or antagonist, and an anti-FASblocking-antibody are both administered to a patient afflicted with adisorder characterized by thrombotic microanglopathy, such as TTP orHUS. Examples of blocking monoclonal antibodies directed against Fasantigen (CD95) are described in International patent applicationpublication number WO 95/10540, hereby incorporated by reference.

[0373] The naturally occurring balance between endogenous stimulatorsand inhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail.

[0374] Unregulated angiogenesis becomes pathologic and sustainsprogression of many neoplastic and non-neoplastic diseases. A number ofserious diseases are dominated by abnormal neovascularization includingsolid tumor growth and metastases, arthritis, some types of eyedisorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech.9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763(1995); Auerbach etal., J. Microvasc. Res. 29:401-411 (1985); Folkman,Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press,New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982);and Folkman et al., Science 221:719-725 (1983). In a number ofpathological conditions, the process of angiogenesis contributes to thedisease state. For example, significant data have accumulated whichsuggest that the growth of solid tumors is dependent on angiogenesis.Folkman and Klagsbrun, Science 235:442-447 (1987).

[0375] The present invention provides for treatment, prevention,diagnosis and/or prognosis of diseases or disorders associated withneovascularization by administration of the DR3 polynucleotides and/orpolypeptides of the invention (including DR3 agonists and/orantagonists). Malignant and metastatic conditions, which can bediagnosed, prognosed, treated and/or prevented with the polynucleotidesand polypeptides of the invention include, but are not limited to thosemalignancies, solid tumors, and cancers described herein and otherwiseknown in the art (for a review of such disorders, see Fishman et al.,Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).

[0376] Additionally, ocular disorders associated with neovascularizationwhich can be diagnosed, prognosed, treated and/or prevented with the DR3polynucleotides and polypeptides of the present invention (including DR3agonists and DR3 antagonists) include, but are not limited to:neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolentalfibroplasia, uveitis, retinopathy of prematurity macular degeneration,corneal graft neovascularization, as well as other eye inflammatorydiseases, ocular tumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

[0377] Additionally, disorders which can be diagnosed, prognosed,treated and/or prevented with the DR3 polynucleotides and polypeptidesof the present invention (including DR3 agonists and DR3 antagonists)include, but are not limited to, hemangioma, arthritis, psoriasis,angiofibroma, atherosclerotic plaques, delayed wound healing,granulations, hemophilic joints, hypertrophic scars, nonunion fractures,Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, andvascular adhesions.

[0378] Polynucleotides and/or polypeptides of the invention, and/oragonists and/or antagonists thereof, are useful in the prognosis,diagnosis, treatment and/or prevention of a wide range of diseasesand/or conditions. Such diseases and conditions include, but are notlimited to, cancer (e.g., immune cell related cancers, breast cancer,prostate cancer, ovarian cancer, follicular lymphoma, glioblastoma,cancer associated with mutation or alteration of p53, brain tumor,bladder cancer, uterocervical cancer, colon cancer, colorectal cancer,non-small cell carcinoma of the lung, small cell carcinoma of the lung,stomach cancer, etc.), lymphoproliferative disorders (e.g.,lymphadenopathy and lymphomas (e.g., EBV induced lymphoproliferationsand Hodgkin's disease), microbial (e.g., viral, bacterial, etc.)infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection(including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7,EBV), adenovirus infection, poxvirus infection, human papilloma virusinfection, hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacterpylori infection, invasive Staphylococcia, etc.), parasitic infection,nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain,cardiovascular disorders (e.g., neovascularization, hypovascularizationor reduced circulation (e.g., ischemic disease (e.g., myocardialinfarction, stroke, etc.)), AIDS, allergy, inflammation,neurodegenerative disease (e.g., Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellardegeneration, etc.), graft rejection (acute and chronic), graft vs. hostdisease, diseases due to osteomyelodysplasia (e.g., aplastic anemia,etc.), joint tissue destruction in rheumatism, liver disease (e.g.,acute and chronic hepatitis, liver injury, and cirrhosis), autoimmunedisease (e.g., multiple sclerosis, myasthenia gravis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, inflammatoryautoimmune diseases, etc.), cardiomyopathy (e.g., dilatedcardiomyopathy), diabetes, diabetic complications (e.g., diabeticnephropathy, diabetic neuropathy, diabetic retinopathy), influenza,asthma, psoriasis, glomerulonephritis, septic shock, and ulcerativecolitis.

[0379] Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof are useful in promotingangiogenesis, wound healing (e.g., wounds, burns, and bone fractures),and regulating bone formation and treating and/or preventingosteoporosis.

[0380] Polynucleotides and/or polypeptides of the invention and/oragonists and/or antagonists thereof are also useful as an adjuvant toenhance immune responsiveness to specific antigen and/or anti-viralimmune responses.

[0381] More generally, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful inregulating (i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment and/or preventionof autoimmune disorders or in the prevention of transplant rejection. Inspecific embodiments, polynucleotides and/or polypeptides of theinvention are used to diagnose, prognose, treat and/or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

[0382] In additional embodiments, DR3 and/or DR3-V1 polynucleotides,polynucleotides and/or other compositions of the invention (e.g., DR3and/or DR3-V1 Fc- or albumin-fusion proteins) are used to diagnose,treat or prevent diseases or conditions associated with allergy and/orinflammation. As demonstrated in Example 15 below, it has been shownthat DR3 interacts with TNF-gamma-beta, a TNF ligand family memberdescribed in detail in International Publication Numbers WO96/14328,WO00/66608, and WO00/08139. TNF-gamma-beta is a proinflammatory moleculeas evidenced by its ability to induce T cell proliferation and secretionof Interferon-gamma and GM-CSF by T cells. TNF-gamma-beta is also ableto enhance an in vivo mixed lymphocyte reaction (MLR) as measured by theparent-into-F1 model of acute graft vs. host disease in which C57BL/6splenic T cells are transferred into (BALB/c×C57BL/6) F1 mice. Thus, theability of DR3 to bind TNF-gamma-beta and to prevent TNF-gamma-betainduced activities (see Example 15) suggests that DR3 and/orDR3-V1polynucleotides and polypeptides are useful as inhibitors ofTNF-gamma-beta function.

[0383] Specifically, DR3 and/or DR3-V1 polynucleotides and polypeptidesand fragments or variants thereof (e.g., soluble forms of DR3 and/orDR3-V1 such as DR3 and/or DR3V-1 Fc- or albumin-fusion proteins) areuseful for the prevention, diagnosis and treatment of inflammationand/or inflammatory diseases and disorders. In particular embodiments,the present invention provides a method of diagnosing, treating,preventing or ameliorating inflammatory diseases or disorders comprisingor alternatively consisting of, administering to an animal, preferably ahuman, in which such diagnosis, treatment, prevention or amelioration isdesired, a DR3 and/or DR3-V1 polynucleotide or polypeptide or fragmentor variant thereof (e.g., soluble forms of DR3 and/or DR3-V1 such as aDR3 and/or DR3-V1 Fc- or albumin-fusion protein) in an amount effectiveto diagnose, treat, prevent or ameliorate the inflammatory disease ordisorder. In specific embodiments, the inflammatory disease or disorderis inflammatory bowel disease. In specific embodiments, the inflammatorydisease or disorder is encephalitis. In specific embodiments, theinflammatory disease or disorder is atherosclerosis. In specificembodiments, the inflammatory disease or disorder is psoriasis. Thepresent invention further provides compositions comprising the DR3and/or DR3-V1 polynucleotide or polypeptide or a fragment or variantthereof (e.g., soluble forms of DR3 and/or DR3-V1 such as a DR3 and/orDR3-V1 Fc- or albumin-fusion protein) and a carrier for use in theabove-described method of diagnosing, treating, preventing orameliorating inflammatory diseases and disorders.

[0384] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating inflammationcomprising or alternatively consisting of, administering to an animal,preferably a human, in which such diagnosis, treatment, prevention oramelioration is desired, a DR3 and/or DR3-V1 polynucleotide orpolypeptide or a fragment or variant thereof (e.g. soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)in an amount effective to diagnose, treat, prevent or ameliorate theinflammation. The present invention further provides compositionscomprising a DR3 and/or DR3-V1 polynucleotide or polypeptide or afragment or variant thereof (e.g., soluble forms of DR3 and/or DR3-V1such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein) and a carrierfor use in the above-described method of diagnosing, treating,preventing or ameliorating inflammation.

[0385] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating graft versus hostdisease (GVHD) comprising or alternatively consisting of, administeringto an animal, preferably a human, in which such diagnosis, treatment,prevention or amelioration is desired, a DR3 and/or DR3-V1polynucleotide or polypeptide or a fragment or variant thereof (e.g.,soluble forms of DR3 and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- oralbumin-fusion protein) in an amount effective to diagnose, treat,prevent or ameliorate the GVHD. The present invention further providescompositions comprising a DR3 and/or DR3-V1 polynucleotide orpolypeptide or a fragment or variant thereof (e.g., soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)and a carrier for use in the above-described method of diagnosing,treating, preventing or ameliorating GVHD.

[0386] In other embodiments, the present invention provides a method ofdiagnosing, treating, preventing or ameliorating autoimmune diseases anddisorders comprising or alternatively consisting of, administering to ananimal, preferably a human, in which such diagnosis, treatment,prevention or amelioration is desired, a DR3 and/or DR3-V1polynucleotide or polypeptide or a fragment or variant thereof (e.g.,soluble forms of DR3 and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- oralbumin-fusion protein) in an amount effective to diagnose, treat,prevent or ameliorate the autoimmune disease or disorder. In specificembodiments, the autoimmune disease or disorder is systemic lupuserythematosus. In specific embodiments, the autoimmune disease ordisorder is arthritis, particularly rheumatoid arthritis. In specificembodiments, the autoimmune disease or disorder is multiple sclerosis.In specific embodiments, the autoimmune disease or disorder is Crohn'sdisease. In specific embodiments, the autoimmune disease or disorder isautoimmune encephalitis. The present invention further providescompositions comprising a DR3 and/or DR3-V1 polynucleotide orpolypeptide or a fragment or variant thereof (e.g., soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)and a carrier for use in the above-described method of diagnosing,treating, preventing or ameliorating autoimmune diseases and disorders.

[0387] In specific embodiments, the present invention provides a methodof diagnosing, treating, preventing or ameliorating allergy or asthmacomprising or alternatively consisting of, administering to an animal,preferably a human, in which such diagnosis, treatment, prevention oramelioration is desired a DR3 and/or DR3-V1 polynucleotide orpolypeptide or a fragment or variant thereof (e.g., soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)or fragment or variant thereof in an amount effective to diagnose,treat, prevent or ameliorate the allergy or asthma. The presentinvention further provides compositions comprising a DR3 and/or DR3-V1polynucleotide or polypeptide or a fragment or variant thereof (e.g.,soluble forms of DR3 and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- oralbumin-fusion protein) and a carrier for use in the above-describedmethod of diagnosing, treating, preventing or ameliorating allergy orasthma.

[0388] The present invention further encompasses methods andcompositions for reducing T cell activation, comprising, oralternatively consisting of, contacting an effective amount of a DR3and/or DR3-V1 polynucleotide or polypeptide or a fragment or variantthereof (e.g., soluble forms of DR3 and/or DR3-V1 such as a DR3 and/orDR3-V1 Fc- or albumin-fusion protein) with cells of hematopoieticorigin, wherein the effective amount of the DR3 and/or DR3-V1polypeptide or a fragment or variant thereof (e.g., soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)reduces T cell activation. In preferred embodiments, the cells ofhematopoietic origin are T cells. In other preferred embodiments, theeffective amount of the DR3 and/or DR3-V1 polypeptide or a fragment orvariant thereof (e.g., soluble forms of DR3 and/or DR3-V1 such as a DR3and/or DR3-V1 Fc- or albumin-fusion protein) reduces TNF-gamma-alphaand/or TNF-gamma-beta induced T cell activation.

[0389] The present invention further encompasses methods andcompositions for reducing T cell activation comprising, or alternativelyconsisting of, administering to an animal, preferably a human, in whichsuch reduction is desired, a DR3 and/or DR3-V1 polynucleotide orpolypeptide or a fragment or variant thereof (e.g., soluble forms of DR3and/or DR3-V1 such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein)or fragment or variant thereof in an amount effective to reduce T cellactivation. The present invention further provides compositionscomprising a DR3 and/or DR3-V1 polynucleotide or polypeptide or afragment or variant thereof (e.g., soluble forms of DR3 and/or DR3-V1such as a DR3 and/or DR3-V1 Fc- or albumin-fusion protein) and a carrierfor use in the above-described method of reducing T cell activation.

[0390] Gene Therapy

[0391] In a specific embodiment, nucleic acids comprising sequencesencoding antibodies or functional derivatives thereof, are administeredto treat, inhibit and/or prevent a disease or disorder associated withaberrant expression and/or activity of a polypeptide of the invention,by way of gene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

[0392] Any of the methods for gene therapy available in the art can beused according to the present invention. Exemplary methods are describedbelow.

[0393] For general reviews of the methods of gene therapy, see Goldspielet al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596(1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIBTECH 11(5):155-215.Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; andKriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, New York.

[0394] In a preferred aspect, the compound comprises nucleic acidsequences encoding an antibody, said nucleic acid sequences being partof expression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both heavy andlight chains, or fragments thereof, of the antibody.

[0395] Delivery of the nucleic acids into a patient may be eitherdirect, in which case the patient is directly exposed to the nucleicacid or nucleic acid-carrying vectors, or indirect, in which case, cellsare first transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

[0396] In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu etal.); WO92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). Alternatively,the nucleic acid can be introduced intracellularly and incorporatedwithin host cell DNA for expression, by homologous recombination (Kollerand Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstraet al., Nature 342:435-438 (1989)).

[0397] In a specific embodiment viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors have been to deleteretroviral sequences that are not necessary for packaging of the viralgenome and integration into host cell DNA. The nucleic acid sequencesencoding the antibody to be used in gene therapy are cloned into one ormore vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdrl gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993).

[0398] Adenoviruses are other viral vectors that can be used in genetherapy. Adenoviruses are especially attractive vehicles for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993), present a reviewof adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994), demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

[0399] Adeno-associated virus (AAV) has also been proposed for use ingene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300(1993); U.S. Pat. No. 5,436,146).

[0400] Another approach to gene therapy involves transferring a gene tocells in tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

[0401] In this embodiment, the nucleic acid is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644(1993);Cline, Pharmac. Ther. 29:69-92 (1985)) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

[0402] The resulting recombinant cells can be delivered to a patient byvarious methods known in the art. Recombinant blood cells (e.g.,hematopoietic stem or progenitor cells) are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art.

[0403] Cells into which a nucleic acid can be introduced for purposes ofgene therapy encompass any desired, available cell type, and include butare not limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asT-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

[0404] In a preferred embodiment, the cell used for gene therapy isautologous to the patient.

[0405] In an embodiment in which recombinant cells are used in genetherapy, nucleic acid sequences encoding an antibody are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see, e.g., PCT Publication WO 94/08598, dated Apr.28, 1994; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth.Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc.61:771 (1986)).

[0406] In a specific embodiment, the nucleic acid to be introduced forpurposes of gene therapy comprises an inducible promoter operably linkedto the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription.

[0407] Modes of Administration

[0408] The invention provides methods of treatment, inhibition andprophylaxis by administration to a subject of an effective amount of acompound or pharmaceutical composition of the invention. In a preferredaspect, the compound is substantially purified (e.g., substantially freefrom substances that limit its effect or produce undesiredside-effects). The subject is preferably an animal, including but notlimited to animals such as cows, pigs, horses, chickens, cats, dogs,etc., and is preferably a mammal, and most preferably human.

[0409] Formulations and methods of administration that can be employedwhen the compound comprises a nucleic acid or an immunoglobulin aredescribed above; additional appropriate formulations and routes ofadministration can be selected from among those described herein below.

[0410] The agonist or antagonists described herein can be administeredin vitro, ex vivo, or in vivo to cells which express the receptor of thepresent invention.

[0411] By administration of an “effective amount” of an agonist orantagonist is intended an amount of the compound that is sufficient toenhance or inhibit a cellular response to a TNF-family ligand andinclude polypeptides. In particular, by administration of an “effectiveamount” of an agonist or antagonists is intended an amount effective toenhance or inhibit DR3-V1 or DR3 mediated apoptosis. Of course, whereapoptosis is to be enhanced, an agonist according to the presentinvention can be co-administered with a TNF-family ligand. One ofordinary skill will appreciate that effective amounts of an agonist orantagonist can be determined empirically and may be employed in pureform or in pharmaceutically acceptable salt, ester or prodrug form. Theagonist or antagonist may be administered in compositions in combinationwith one or more pharmaceutically acceptable excipients.

[0412] It will be understood that, when administered to a human patient,the total daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

[0413] As a general proposition, the total pharmaceutically effectiveamount of a DR3 polypeptide administered parenterally per dose will bein the range of about 1 μg/kg/day to 10 mg/kg/day of patient bodyweight, although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the DR3 agonists or antagonists istypically administered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

[0414] Dosing may also be arranged in a patient specific manner toprovide a predetermined concentration of an agonist or antagonist in theblood, as determined by the RIA technique. Thus patient dosing may beadjusted to achieve regular on-going trough blood levels, as measured byRIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.

[0415] Various delivery systems are known and can be used to administera compound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

[0416] Pharmaceutical compositions are provided comprising an agonist orantagonist and a pharmaceutically acceptable carrier or excipient, whichmay be administered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray.Importantly, by co-administering an agonist and a TNF-family ligand,clinical side effects can be reduced by using lower doses of both theligand and the agonist. It will be understood that the agonist can be“co-administered” either before, after, or simultaneously with theTNF-family ligand, depending on the exigencies of a particulartherapeutic application. By “pharmaceutically acceptable carrier” ismeant a non-toxic solid, semisolid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. The term“parenteral” as used herein refers to modes of administration, whichinclude intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

[0417] Pharmaceutical compositions of the present invention forparenteral injection can comprise pharmaceutically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsionsas well as sterile powders for reconstitution into sterile injectablesolutions or dispersions just prior to use.

[0418] In addition to soluble DR3-V1 or DR3 polypeptides, DR3-V1 or DR3polypeptide containing the transmembrane region can also be used whenappropriately solubilized by including detergents, such as CHAPS orNP-40, with buffer.

[0419] In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

[0420] In another embodiment, the compound or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

[0421] In yet another embodiment, the compound or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., 1983, Macromol. Sci.Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the therapeutic target, i.e., thebrain, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

[0422] Other controlled release systems are discussed in the review byLanger (1990, Science 249:1527-1533).

[0423] In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

[0424] The present invention also provides pharmaceutical compositions.Such compositions comprise a therapeutically effective amount of acompound, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

[0425] The compounds of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

[0426] The amount of the compound of the invention which will beeffective in the treatment, inhibition and/or prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

[0427] For antibodies, the dosage administered to a patient is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, thedosage administered to a patient is between 0.1 mg/kg and 20 mg/kg ofthe patient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto thelimmune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

[0428] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0429] The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

[0430] The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

[0431] In one embodiment, the compositions of the invention areadministered in combination with other members of the TNF family. TNF,TNF-related or TNF-like molecules that may be administered with thecompositions of the invention include, but are not limited to, solubleforms of TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (foundin complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-γ (International Publication No. WO 96/14328),TNF-γ-α (International Publication No. WO 00/08139), TNF-γ-β(International Publication No. WO 00/08139), AIM-I (InternationalPublication No. WO 97/33899), AIM-II (International Publication No. WO97/34911), endokine-α (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-α(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms of CD 154, CD70, and CD153.

[0432] In another embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g., agonisticor antagonistic antibodies).

[0433] In yet another embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, or more ofthe following compositions: tacrolimus (Fujisawa), thalidomide (e.g.,Celgene), anti-Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab(Biotest), MAK-195F (Knoll), ASM-981 (Novartis), interleukin-1 receptor(e.g., Immunex), interleukin-4 receptor (e.g., Immunex), ICM3 (ICOS),BMS-188667 (Bristol-Myers Squibb), anti-TNF Ab (e.g., Therapeuticantibodies), CG-1088 (Celgene), anti-B7 Mab (e.g., Innogetics), MEDI-507(BioTransplant), ABX-CBL (Abgenix).

[0434] In certain embodiments, compositions of the invention areadministered in combination with antiretroviral agents, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat, prevent, and/or diagnoseAIDS and/or to treat, prevent, and/or diagnose HIV infection.

[0435] In other embodiments, compositions of the invention may beadministered in combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with ISONIAZD™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™to prophylactically treat, prevent, and/or diagnose an opportunisticMycobacterium avium complex infection. In another specific embodiment,compositions of the invention are used in any combination withRIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, compositions ofthe invention are used in any combination with GANCICLOVIR™, FOSCARNET™,and/or CIDOFOVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic cytomegalovirus infection. In another specific embodiment,compositions of the invention are used in any combination withFLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic fungal infection. Inanother specific embodiment, compositions of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat, prevent, and/or diagnose an opportunistic herpes simplex virustype I and/or type II infection. In another specific embodiment,compositions of the invention are used in any combination withPYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat, prevent,and/or diagnose an opportunistic Toxoplasma gondii infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat,prevent, and/or diagnose an opportunistic bacterial infection.

[0436] In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

[0437] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, β-lactam(glycopeptide), β-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

[0438] Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

[0439] In specific embodiments, compositions of the invention areadministered in combination with immunosuppressants. Immunosuppressantspreparations that may be administered with the compositions of theinvention include, but are not limited to, ORTHOCLONE™ (OKT3),SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™ (tacrolimus),CELLCEPT™ (mycophenolate), Azathioprine, glucorticosteroids, andRAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants maybe used to prevent rejection of organ or bone marrow transplantation.

[0440] In one embodiment, the compositions of the invention areadministered in combination with steroid therapy. Steroids that may beadministered in combination with the compositions of the invention,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, compositions of the invention are administered incombination with prednisone. In a further specific embodiment, thecompositions of the invention are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the compositions of the invention andprednisone are those described herein, and include, but are not limitedto, azathioprine, cyclophosphamide, and cyclophosphamide IV. In anotherspecific embodiment, compositions of the invention are administered incombination with methylprednisolone. In a further specific embodiment,the compositions of the invention are administered in combination withmethylprednisolone and an immunosuppressive agent. Immunosuppressiveagents that may be administered with the compositions of the inventionand methylprednisolone are those described herein, and include, but arenot limited to, azathioprine, cyclophosphamide, and cyclophosphamide IV.

[0441] In another embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

[0442] In one embodiment, the compositions of the invention areadministered in combination with an NSAID.

[0443] In another embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, ten, ormore of the following drugs: NRD-101 (Hoechst Marion Roussel),diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin(Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000(Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-1Ra genetherapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech),DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNFreceptor 1 (synergen; Amgen), IPR-6001 (Institute for PharmaceuticalResearch), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals),BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim),LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), andbutixocort propionate (WarnerLambert).

[0444] In yet another embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, and prednisolone. In a morepreferred embodiment, the compositions of the invention are administeredin combination with an antimalarial, methotrexate, anti-TNF antibody,ENBREL™ and/or suflasalazine. In one embodiment, the compositions of theinvention are administered in combination with methotrexate. In anotherembodiment, the compositions of the invention are administered incombination with anti-TNF antibody. In another embodiment, thecompositions of the invention are administered in combination withmethotrexate and anti-TNF antibody. In another embodiment, thecompositions of the invention are administered in combination withsuflasalazine. In another specific embodiment, the compositions of theinvention are administered in combination with methotrexate, anti-TNFantibody, and suflasalazine. In another embodiment, the compositions ofthe invention are administered in combination ENBREL™. In anotherembodiment, the compositions of the invention are administered incombination with ENBREL™ and methotrexate. In another embodiment, thecompositions of the invention are administered in combination withENBREL™, methotrexate and suflasalazine. In another embodiment, thecompositions of the invention are administered in combination withENBREL™, methotrexate and suflasalazine. In other embodiments, one ormore antimalarials is combined with one of the above-recitedcombinations. In a specific embodiment, the compositions of theinvention are administered in combination with an antimalarial (e.g.,hydroxychloroquine), ENBREL™, methotrexate and suflasalazine. In anotherspecific embodiment, the compositions of the invention are administeredin combination with an antimalarial (e.g., hydroxychloroquine),sulfasalazine, anti-TNF antibody, and methotrexate.

[0445] In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,β-lactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones,cephalosporins, erythromycin, ciprofloxacin, and streptomycin.

[0446] In an additional embodiment, the compositions of the inventionare administered alone or in combination with an anti-inflammatoryagent. Anti-inflammatory agents that may be administered with thecompositions of the invention include, but are not limited to,glucocorticoids and the nonsteroidal anti-inflammatories,aminoarylcarboxylic acid derivatives, arylacetic acid derivatives,arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acidderivatives, pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine,3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone,nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime,proquazone, proxazole, and tenidap.

[0447] In another embodiment, compositions of the invention areadministered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the compositionsof the invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0448] In an additional embodiment, the compositions of the inventionare administered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,IL-15, anti-CD40, CD40L, IFN-γ and TNF-α.

[0449] In an additional embodiment, the compositions of the inventionare administered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PIGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above-mentioned references are incorporated herein byreference herein.

[0450] In an additional embodiment, the compositions of the inventionare administered in combination with Fibroblast Growth Factors.Fibroblast Growth Factors that may be administered with the compositionsof the invention include, but are not limited to, FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12,FGF-13, FGF-14, and FGF-15.

[0451] In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

[0452] The invention also provides a method of delivering compositionscontaining the polypeptides of the invention (e.g., compositionscontaining DR3 polypeptides or anti-DR3 antibodies associated withheterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs) to targeted cells, expressing the membrane-bound form of DR3on their surface, or alternatively, a DR3 receptor on their surface. DR3polypeptides or anti-DR3 antibodies of the invention may be associatedwith heterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions.

[0453] In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (e.g., DR3 or anti-DR3antibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

[0454] In another embodiment, the invention provides a method for thespecific destruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., DR3 polypeptides oranti-DR3 antibodies) in association with toxins or cytotoxic prodrugs.

[0455] In a specific embodiment, the invention provides a method for thespecific destruction of cells expressing the membrane-bound form of DR3on their surface (e.g., spleen, bone marrow, kidney and PBLs) byadministering anti-DR3 antibodies in association with toxins orcytotoxic prodrugs.

[0456] By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, cytotoxins (cytotoxic agents), or anymolecules or enzymes not normally present in or on the surface of a cellthat under defined conditions cause the cell's death. Toxins that may beused according to the methods of the invention include, but are notlimited to, radioisotopes known in the art, compounds such as, forexample, antibodies (or complement fixing containing portions thereof)that bind an inherent or induced endogenous cytotoxic effector system,thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,pokeweed antiviral protein, α-sarcin and cholera toxin. “Toxin” alsoincludes a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., α-emitters such as, for example, ²¹³Bi, orother radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

[0457] Techniques known in the art may be applied to label proteins(including antibodies) of the invention. Such techniques include, butare not limited to, the use of bifunctional conjugating agents (see,e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;4,994,560; and 5,808,003; the contents of each of which are herebyincorporated by reference in its entirety). A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0458] By “cytotoxic prodrug” is meant a non-toxic compound that isconverted by an enzyme, normally present in the cell, into a cytotoxiccompound. Cytotoxic prodrugs that may be used according to the methodsof the invention include, but are not limited to, glutamyl derivativesof benzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubisin, andphenoxyacetamide derivatives of doxorubicin.

[0459] As discussed in more detail below, the invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention. Optionally associated with such container(s) can be a noticein the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

[0460] Diagnosis and Imaging

[0461] Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

[0462] The invention provides a diagnostic assay for diagnosing adisorder, comprising (a) assaying the expression of the polypeptide ofinterest in cells or body fluid of an individual using one or moreantibodies specific to the polypeptide interest and (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a particulardisorder. With respect to cancer, the presence of a relatively highamount of transcript in biopsied tissue from an individual may indicatea predisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0463] Antibodies of the invention can be used to assay protein levelsin a biological sample using classical immunohistological methods knownto those of skill in the art (e.g., see Jalkanen, M. et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M. et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

[0464] One aspect of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof the interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); c) determining background level; and d) detecting thelabeled molecule in the subject, such that detection of labeled moleculeabove the background level indicates that the subject has a particulardisease or disorder associated with aberrant expression of thepolypeptide of interest. Background level can be determined by variousmethods including, comparing the amount of labeled molecule detected toa standard value previously determined for a particular system.

[0465] It will be understood in the art that the size of the subject andthe imaging system used will determine the quantity of imaging moietyneeded to produce diagnostic images. In the case of a radioisotopemoiety, for a human subject, the quantity of radioactivity injected willnormally range from about 5 to 20 millicuries of 99mTc. The labeledantibody or antibody fragment will then preferentially accumulate at thelocation of cells that contain the specific protein. In vivo tumorimaging is described in S. W. Burchiel et al., “Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

[0466] Depending on several variables, including the type of label usedand the mode of administration, the time interval following theadministration for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject and for unbound labeled molecule tobe cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to12 hours. In another embodiment the time interval followingadministration is 5 to 20 days or 5 to 10 days.

[0467] In an embodiment, monitoring of the disease or disorder iscarried out by repeating the method for diagnosing the disease ordisease, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

[0468] Presence of the labeled molecule can be detected in the patientusing methods known in the art for in vivo scanning. These methodsdepend upon the type of label used. Skilled artisans will be able todetermine the appropriate method for detecting a particular label.Methods and devices that may be used in the diagnostic methods of theinvention include, but are not limited to, computed tomography (CT),whole body scan such as position emission tomography (PET), magneticresonance imaging (MRI), and sonography.

[0469] In a specific embodiment, the molecule is labeled with aradioisotope and is detected in the patient using a radiation responsivesurgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). Inanother embodiment, the molecule is labeled with a fluorescent compoundand is detected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

[0470] Kits

[0471] The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope that isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody that does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

[0472] In another specific embodiment of the present invention, the kitis a diagnostic kit for use in screening serum containing antibodiesspecific against proliferative and/or cancerous polynucleotides andpolypeptides. Such a kit may include a control antibody that does notreact with the polypeptide of interest. Such a kit may include asubstantially isolated polypeptide antigen comprising an epitope that isspecifically immunoreactive with at least one anti-polypeptide antigenantibody. Further, such a kit includes means for detecting the bindingof said antibody to the antigen (e.g., the antibody may be conjugated toa fluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

[0473] In a more specific embodiment the detecting means of theabove-described kit includes a solid support to which said polypeptideantigen is attached. Such a kit may also include a non-attachedreporter-labeled anti-human antibody. In this embodiment, binding of theantibody to the polypeptide antigen can be detected by binding of thesaid reporter-labeled antibody.

[0474] In an additional embodiment, the invention includes a diagnostickit for use in screening serum, containing antigens of the polypeptideof the invention. The diagnostic kit includes a substantially isolatedantibody specifically immunoreactive with polypeptide or polynucleotideantigens, and means for detecting the binding of the polynucleotide orpolypeptide antigen to the antibody. In one embodiment, the antibody isattached to a solid support. In a specific embodiment, the antibody maybe a monoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

[0475] In one diagnostic configuration, test serum is reacted with asolid phase reagent having a surface-bound antigen obtained by themethods of the present invention. After binding with specific antigenantibody to the reagent and removing unbound serum components bywashing, the reagent is reacted with reporter-labeled anti-humanantibody to bind reporter to the reagent in proportion to the amount ofbound anti-antigen antibody on the solid support. The reagent is againwashed to remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme, which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

[0476] The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

[0477] Thus, the invention provides an assay system or kit for carryingout this diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

EXAMPLE 1

[0478] Expression and Purification in E. coli

[0479] The DNA sequence encoding the mature DR3-V1 protein in the cDNAcontained in ATCC No. 97456 is amplified using PCR oligonucleotideprimers specific to the amino terminal sequences of the DR3-V1 proteinand to vector sequences 3′ to the gene. Additional nucleotidescontaining restriction sites to facilitate cloning are added to the 5′and 3′ sequences respectively.

[0480] The following primers are used for expression of DR3extracellular domain in E. coli. The 5′ primer:5′-GCGCCATGGGGGCCCGGCGGCAG-3′ (SEQ ID NO:7), contains an NcoI site and15 nucleotide starting from 290 nucleotide to 304 in SEQ ID NO: 1. The3′ primer: 5′-GCGAAGCTTCTAGGACCCAGAACATCTGCC-3′ (SEQ ID NO:8), containsa HindIII site, a stop codon and 18 nucleotides complimentary tonucleotides from 822 to 840 in SEQ ID NO:1. Vector is pQE60. The proteinis not tagged.

[0481] The restriction sites are convenient to restriction enzyme sitesin the bacterial expression vector pQE60, which are used for bacterialexpression in these examples. (Qiagen, Inc. 9259 Eton Avenue,Chatsworth, Calif., 91311). pQE60 encodes ampicillin antibioticresistance (“Amp^(r)”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”).

[0482] The amplified DR3-V 1 DNA and the vector pQE60 both are digestedwith NcoI and HindIII and the digested DNAs are then ligated together.Insertion of the DDCR protein DNA into the restricted pQE60 vectorplaces the DR3-V1 protein coding region downstream of and operablylinked to the vector's IPTG-inducible promoter and in-frame with aninitiating AUG appropriately positioned for translation of DR3-V1protein.

[0483] The ligation mixture is transformed into competent E. coli cellsusing standard procedures. Such procedures are described in Sambrook etal., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance (“Kan^(r)”), isused in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingDR3-V1 protein, is available commercially from Qiagen.

[0484] Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis.

[0485] Clones containing the desired constructs are grown overnight(“O/N”) in liquid culture in LB media supplemented with both ampicillin(100 μg/ml) and kanamycin (25 μg/ml).

[0486] The O/N culture is used to inoculate a large culture, at adilution of approximately 1:100 to 1:250. The cells are grown to anoptical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies into 8M Urea. The 8M Urea solution containing the solubilizedprotein is passed over a PD-10 column in 2× phosphate-buffered saline(“PBS”), thereby removing the urea, exchanging the buffer and refoldingthe protein. The protein is purified by a further step of chromatographyto remove endotoxin. Then, it is sterile filtered. The sterile filteredprotein preparation is stored in 2× PBS at a concentration of 95μ/ml.

EXAMPLE 2

[0487] Expression in Mammalian Cells

[0488] Most of the vectors used for the transient expression of a givengene sequence in mammalian cells carry the SV40 origin of replication.This allows the replication of the vector to high copy numbers in cells(e.g., COS cells) that express the T antigen required for the initiationof viral DNA synthesis. Any other mammalian cell line can also beutilized for this purpose.

[0489] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV).However, also cellular signals can be used (e.g., human actin promoter).Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI(ATCC67109). Mammalian host cells that could be used include, humanHeLa, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos7, and CV1 African green monkey cells, quail QC1-3 cells, mouse L cells,and Chinese hamster ovary (CHO) cells.

[0490] Alternatively, a gene of interest can be expressed in stable celllines that contain the gene integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

[0491] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase) is auseful marker to develop cell lines that carry several hundred or evenseveral thousand copies of the gene of interest. Using this marker, themarnmalian cells are grown in increasing amounts of methotrexate forselection and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) cells are often used for the production ofproteins.

[0492] The expression vectors pCl and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology 438:44701 (March 1985)), plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

EXAMPLE 2A

[0493] Expression of Extracellular Soluble Domain of DR3-V1 and DR3 inCOS Cells

[0494] The expression plasmid, pDR3-V1 HA, is made by cloning a cDNAencoding DR3-V1 (ATCC No. 97456) into the expression vector pcDNA/Amp(which can be obtained from Invitrogen, Inc.). Expression plasmid, pDR3HA, is made by cloning a cDNA encoding DR3 (ATCC No.97757) into theexpression vector pcDNAI/Amp.

[0495] The expression vector pcDNAI/amp contains: (1) an E. coli originof replication effective for propagation in E. coli and otherprokaryotic cell; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron, and a polyadenylation signal arranged so that a cDNAconveniently can be placed under expression control of the CMV promoterand operably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker.

[0496] A DNA fragment encoding the entire DR3-V1 or Dr3 precursor and aHA tag fused in frame to its 3′ end is cloned into the polylinker regionof the vector so that recombinant protein expression is directed by theCMV promoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein described by Wilson et al., Cell 37:767(1984). The fusion of the HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0497] The plasmid construction strategy is as follows:

[0498] The DR3-V1 or DR3 cDNA of the deposit cDNA is amplified usingprimers that contained convenient restriction sites, much as describedabove regarding the construction of expression vectors for expression ofDR3-V1 or DR3 in E. coli and S. frugiperda.

[0499] To facilitate detection, purification and characterization of theexpressed DR3-V1 or DR3, one of the primers contains a hemagglutinin tag(“HA tag”) as described above.

[0500] Suitable primers for DR3-V1 include the following, which are usedin this example, the 5′ primer: 5′ CGCGGATCCGCCATCATGGAGGAGACGCAGCAG 3′(SEQ ID NO:9) contains the underlined BamHI site, an ATG start codon and5 codons thereafter.

[0501] Suitable primers for DR3 include the following, which are used inthis example, the 5′ primer: 5′ CGCGGATCCGCCATCATGGAGCAGCGGCCGCGG 3′(SEQ ID NO:10) contains the underlined BamHI site, an ATG start codonand 5 codons thereafter.

[0502] The 3′ primer for both DR3 and DR3-V1, containing the underlinedXbaI site, stop codon, hemagglutinin tag and last 14 nucleotide of 3′coding sequence (at the 3′ end), has the following sequence: (SEQ IDNO:11) 5′GCGTCTAGATCAAAGCGTAGTCTGGGACGTCGTATGGG TACGGGCCGCGCTGCA 3′.

[0503] The PCR amplified DNA fragment and the vector, pcDNAI/Amp, aredigested with BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037) thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisand gel sizing for the presence of the DR3-V1 or DR3-encoding fragment.

[0504] For expression of recombinant DR3-V 1 or DR3, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989).

[0505] Cells are incubated under conditions for expression of DR3-V1 orDR3 by the vector.

[0506] Expression of the DR3-V1 HA fusion protein or the DR3 HA fusionprotein is detected by radiolabelling and immunoprecipitation, usingmethods described in, for example Harlow et al., Antibodies: aLaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1988). To this end, two days after transfection,the cells are labeled by incubation in media containing ³⁵S-cysteine for8 hours. The cells and the media are collected, and the cells are washedand then lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1%NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described byWilson et al. cited above. Proteins are precipitated from the celllysate and from the culture media using an HA-specific monoclonalantibody. The precipitated proteins are analyzed by SDS-PAGE gels andautoradiography. An expression product of the expected size is seen inthe cell lysate, which is not seen in negative controls.

EXAMPLE 2B

[0507] Expression and Purification of Human DR3-V1 and DR3 Using the CHOExpression System

[0508] The vector pC1 is used for the expression of DR3-V1 or DR3 (ATCCNo. 97456 or ATCC No.97757, respectively) protein. Plasmid pC1 is aderivative of the plasmid pSV2-dhfr (ATCC Accession No.37146). Bothplasmids contain the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., F. W. Alt, et al., J Biol.Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys.Acta, 1097:107-143 (1990); M. J. Page and M. A. Sydenham, Biotechnology9:64-68 (1991)). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

[0509] Plasmid pC1 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rous SarcomaVirus (Cullen et al., Molecular and Cellular Biology 5:438-447 (March1985)), plus a fragment isolated from the enhancer of the immediateearly gene of human cytomegalovirus (CMV) (Boshart etal., Cell41:521-530(1985)). Downstream from the promoter are the following singlerestriction enzyme cleavage sites that allow the integration of thegenes: BamHI followed by the 3′ intron and the polyadenylation site ofthe rat preproinsulin gene. Other high efficient promoters can also beused for the expression, e.g., the human β-actin promoter, the SV40early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNAother signals, e.g., from the human growth hormone or globin genes canbe used as well.

[0510] Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0511] The plasmid pC1 is digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0512] The DNA sequence encoding DR3-V1 or DR3 in the deposited cDNA isamplified using PCR oligonucleotide primers specific to the amino acidcarboxyl terminal sequence of the DR3-V1 or DR3 protein and to vectorsequences 3′ to the gene. Additional nucleotides containing restrictionsites to facilitate cloning are added to the 5′ and 3′ sequencesrespectively.

[0513] The 5′ oligonucleotide primer for DR3-V1 has the sequence: 5′CGCGGATCC GCCATCATGGAGGAGACGCAGCAG 3′ (SEQ ID NO:12) containing theunderlined BamHI restriction site, which encodes a start AUG, followedby the Kozak sequence and 18 nucleotides of the DR3-V1 coding sequenceset out in SEQ ID NO:1 beginning with the first base of the ATG codon.

[0514] The 5′ oligonucleotide primer for DR3 has the sequence: 5′CGCGGATCCGCC ATCATGGAGCAGCGGCCGCGG 3′ (SEQ ID NO: 13) containing theunderlined BamHI restriction site, which encodes a start AUG, followedby the Kozak sequence and 18 nucleotides of the DR3 coding sequence setout in SEQ ID NO:3 beginning with the first base of the ATG codon.

[0515] The 3′ primer for both DR3 and DR3-V1 has the sequence: 5′CGCGGATCCTCACGGGCCGCGCTGCA 3′ (SEQ ID NO: 14) containing the underlinedBamHI restriction site followed by 17 nucleotides complementary to thelast 14 nucleotides of the DR3-V 1 or DR3 coding sequence set out in SEQID NO:1 or SEQ ID NO:3, respectively, plus the stop codon.

[0516] The restrictions sites are convenient to restriction enzyme sitesin the CHO expression vectors pC1.

[0517] The amplified DR3 orDR3-V1 DNA and the vector pC1 both aredigested with BamHI and the digested DNAs then ligated together.Insertion of the DR3-V1 or DR3 DNA into the BamHI restricted vectorplaced the DR3-V1 or DR3 coding region downstream of and operably linkedto the vector's promoter. The sequence of the inserted gene is confirmedby DNA sequencing.

[0518] Transfection of CHO-DHFR-Cells

[0519] Chinese hamster ovary cells lacking an active DHFR enzyme areused for transfection. 5 μg of the expression plasmid C1 arecotransfected with 0.5 μg of the plasmid pSVneo using the lipofectionmethod (Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated from 10-14 days. After this period,single clones are trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25 nM, 50 nM, 100 nM,200 nM, 400 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). Thesame procedure is repeated until clones grow at a concentration of 100μM.

[0520] The expression of the desired gene product is analyzed by Westernblot analysis and SDS-PAGE.

EXAMPLE 3

[0521] Cloning and Expression of the Soluble Extracellular Domain ofDR3-V1 and DR3 in a Baculovirus Expression System

[0522] The cDNA sequence encoding the soluble extracellular domain ofDR3-V1 or DR3 protein in the deposited clone (ATCC No. 97456 or ATCC No.97757, respectively) is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene.

[0523] The 5′ primer for DR3-V1 has the sequence: 5′CGCGGATCCGCCATCATGGA GGAGACGCAGCAG 3′ (SEQ ID NO:15) containing theunderlined BamHI restriction enzyme site followed by a Kozak sequenceand a number of bases of the sequence of DR3-V1 of SEQ ID NO:1. Insertedinto an expression vector, as described below, the 5′ end of theamplified fragment encoding DR3-V1 provides an efficient signal peptide.An efficient signal for initiation of translation in eukaryotic cells,as described by M. Kozak, J. Mol. Biol. 196:947-950 (1987) isappropriately located in the vector portion of the construct.

[0524] The 5′ primer for DR3 has the sequence: 5′CGCGGATCCGCCATCATGGAGCA GCGGCCGCGG 3′ (SEQ ID NO:16) containing theunderlined BamHI restriction enzyme site followed by a Kozak sequenceand a number of bases of the sequence of DR3 of SEQ ID NO:3. Insertedinto an expression vector, as described below, the 5′ end of theamplified fragment encoding DR3 provides an efficient signal peptide. Anefficient signal for initiation of translation in eukaryotic cells, asdescribed by M. Kozak, J. Mol. Biol. 196:947-950 (1987) is appropriatelylocated in the vector portion of the construct.

[0525] The 3′ primer for both DR3 and DR3-V1 has the sequence: 5′GCGAGATCTAGT CTGGACCCAGAACATCTGCCTCC 3′ (SEQ ID NO: 17) containing theunderlined XbaI restriction followed by nucleotides complementary to theDR3-V1 or DR3 nucleotide sequence set out in SEQ ID NO:1 or SEQ ID NO:3,respectively, followed by the stop codon.

[0526] The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.)The fragment then is digested with BamHI and Asp718 and again ispurified on a 1% agarose gel. This fragment is designated herein F2.

[0527] The vector pA2 is used to express the DR3-V1 or DR3 protein inthe baculovirus expression system, using standard methods, such as thosedescribed in Summers et al., A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987). This expression vector contains thestrong polyhedron promoter of the Autograph califomica nuclearpolyhedrosis virus (ACMNPV) followed by convenient restriction sites.For an easy selection of recombinant virus the β-galactosidase gene fromE. coli is inserted in the same orientation as the polyhedron promoterand is followed by the polyadenylation signal of the polyhedron gene.The polyhedron sequences are flanked at both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that express the cloned polynucleotide.

[0528] Many other baculovirus vectors could be used in place of pA2,such as pAc373, pVL941 and pAcIM1 provided, as those of skill readilywill appreciate, that construction provides appropriately locatedsignals for transcription, translation, trafficking and the like, suchas an in-frame AUG and a signal peptide, as required. Such vectors aredescribed in Luckow et al., Virology 170:31-39 (1989), among others.

[0529] The plasmid is digested with the restriction enzymes BamHI andXbaI and then is dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.). This vector DNA is designated herein “V2”.

[0530] Fragment F2 and the dephosphorylated plasmid V2 are ligatedtogether with T4 DNA ligase. E. coli HB101 cells are transformed withligation mix and spread on culture plates. Bacteria are identified thatcontain the plasmid with the human DDCR gene by digesting DNA fromindividual colonies using BamHI and XbaI and then analyzing thedigestion product by gel electrophoresis. The sequence of the clonedfragment is confirmed by DNA sequencing. This plasmid is designatedherein pBac DR3-V1 or pBac DR3.

[0531] 5 μg of the plasmid pBac DR3-V1 or pBac DR3 is co-transfectedwith 1.0 μg of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg ofthe plasmid pBac DR3-V1 are mixed in a sterile well of a microliterplate containing 50 μl of serum free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27° C. After 5 hours the ttansfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27° C. for four days.

[0532] After four days the supernatant is collected and a plaque assayis performed, as described by Summers and Smith, cited above. An agarosegel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used toallow easy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10).

[0533] Four days after serial dilution, the virus is added to the cells.After appropriate incubation, blue stained plaques are picked with thetip of an Eppendorf pipette. The agar containing the recombinant virusesis then resuspended in an Eppendorf tube containing 200 μl of Grace'smedium. The agar is removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. A clonecontaining properly inserted DR3-V1 or DR3 is identified by DNA analysisincluding restriction mapping and sequencing. This is designated hereinas V DR3-V1 or V-DR3.

[0534] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-DR3-V1 at a multiplicity of infection (“MOI”) of about 2(about 1 to about 3). Six hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation, lysed and the labeled proteins arevisualized by SDS-PAGE and autoradiography.

EXAMPLE 4

[0535] Tissue Distribution of DR3-V1 Gene Expression

[0536] Northern blot analysis is carried out to examine DR3-V1 gene(ATCC No. 97456) expression in human tissues, using methods describedby, among others, Sambrook et al., cited above. A cDNA probe containingthe entire nucleotide sequence of the DR3-V1 protein (SEQ ID NO: 1) islabeled with ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for DR3-V1 mRNA.

[0537] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures. Expression of DR3-V1 was detected intissues enriched in lymphocytes including peripheral blood leukocytes(PBLs), thymus, spleen, colon, and small intestine. DR3-V1 expressionappears to be restricted to lymphocyte compartments, it can be envisagedthat DR3-V1 plays a role in lymphocyte homeostasis.

[0538] Tissue Distribution of DR3 Gene Expression

[0539] Northern blot analysis is carried out to examine DR3 gene (ATCCNo. 97757) expression in human tissues, using methods described by,among others, Sambrook et al., cited above. A cDNA probe containing theentire nucleotide sequence of the DR3 protein (SEQ ID NO:1) is labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for DR3 mRNA.

[0540] Multiple Tissue Northern (MTN) blots containing various humantissues (H) or human immune system tissues (IM) are obtained fromClontech and are examined with labeled probe using ExpressHyb™hybridization solution (Clontech) according to manufacturer's protocolnumber PT1190-1. Following hybridization and washing, the blots aremounted and exposed to film at −70° C. overnight, and films developedaccording to standard procedures.

[0541] Expression of DR3 was detected in tissues enriched in lymphocytesincluding peripheral blood leukocytes (PBLs), thymus, spleen, colon, andsmall intestine. By contrast, TNFR-1 is ubiquitously expressed andFas/APO-1 is expressed in lymphocytes, liver, heart, lung, kidney, andovary (Watanabe-Fukunaga et al., J. Immunol 148:1274-9 (1992)).

[0542] DR3 expression appears to be restricted to lymphocytecompartments, it can be envisaged that DR3 plays a role in lymphocytehomeostasis.

[0543] Northern Blot Analysis of DR3 in Various Cell Lines

[0544] Methods

[0545] Cells

[0546] Unless stated otherwise, cell lines were obtained from theAmerican Type Culture Collection (Manassas, Va.). The myeloid (Koeffleret al. (1980); Koeffler (1983); Harris and Ralph (1985); and Tucker etal. (1987)) and B-cell lines (Jonak et al. (1922)) studied representcell types at different stages of the differentiation pathway. KG1a andPLB 985 cells (Tucker et al. (1987)) were obtained from H. P. Koeffler(UCLA School of Medicine). BJA-B was from Z. Jonak (SmithKline Beecham).TF274, a stromal cell line exhibiting osteoblastic features, wasgenerated from the bone marrow of a healthy male donor (Z. Jonak and K.B. Tan, unpublished). Primary carotid artery endothelial cells werepurchased from Clonetics Corp. (San Diego, Calif.) and monocytes wereprepared by differential centrifugation of peripheral blood mononuclearcells and adhesion to tissue culture dish. CD19+, CD4+ and CD8+ cells(>90% pure) were isolated with cell type specific immunomagnetic beads(Drynal, Lake Success, N.Y.).

[0547] RNA Analysis

[0548] Total RNA of adult tissues were purchased from Clonetech (PaloAlto, Calif.). Total RNA was extracted from cell lines (in exponentialgrowth phase) and primary cells with TriReagent (Molecular ResearchCenter, Inc., Cincinnati, Ohio). 5 to 7.5 μg of total RNA wasfractionated in a 1% agarose gel containing formaldehyde cast in a WideMini-Sub Cell gel tray (Bio-Rad, Hercules, Calif.) as described(Sambrook, et al.) with slight modifications. The formaldehydeconcentration was reduced to 0.5M and the RNA was stained prior toelectrophoresis with 100 μg/ml of ethidium bromide that was added to theloading buffer. After electrophoresis with continuous bufferrecirculation (60 volts/90 min), the gel was photographed and the RNAwas transferred quantitatively to Zeta-probe nylon membrane (Biorad,Hercules, Calif.) by vacuum-blotting with 25 mM NaOH for 90 min. Afterneutralization for 5-10 min, with 1M Tris-HCl, pH 7.5 containing 3MNaCl, the blots were prehybridized with 50% formamide, 8% dextransulfate, 6×SSPE, 0.1% SDS and 100 μg/ml of sheared and denatured salmonsperm DNA for at least 30 min at 42° C. cDNA inserts labeled with³²P-dCTP by random priming (Stratagene, La Jolla, Calif.), weredenatured with 0.25M NaOH (10 min at 37° C.) and added to theprehybridization solution. After 24-65 hr at 42° C., the blots werewashed under high stringency conditions (Sambrook, et al.) and exposedto X-ray films.

[0549] Results

[0550] Expression of DR3 was assessed by Northern blot in the followingcell lines: TF274 (bone marrow stromal); MG63, TE85 (osteosarcoma); K562(erythroid); KG1a, KG1, PLB985, HL60, U937, TNHP-1 (myeloid); REH, BJAB,Raji, IM-9 (B cell); Sup-T1, Jurkat, H9, Molt-3 (T cell); RL95-2(endometrial carcinoma); MCF-7 (breast cancer); BE, HT29 (colon cancer);IMR32 (neuroblastoma) and could only be detected in KG1a cells. DR3expression was detected in several lymphoblast cell lines. In thepurified human hematopoietic cell populations, DR3 was weakly expressedin CD 19+ cells, and more highly expressed in monocytes. However thehighest levels were observed in T cells (CD4+ or CD8+) upon stimulationwith PMA and PHA, indicating that DR3 probably plays a role in theregulation of T cell activation.

EXAMPLE 5

[0551] Intracellular Signaling Molecules used by DR3 Protein

[0552] In vitro and in vivo binding studies were undertaken toinvestigate DR3 signaling pathways. Since DR3 contains a death domain,the inventors postulated that DR3, like TNFR-1 and Fas/APO-1, maytransduce signals by recruiting death domain-containing adaptermolecules (DAMs) such as FADD, TRADD, and RIP.

[0553] Experimental Design

[0554] In vitro binding experiments were performed as describedpreviously (A. M. Chinnaiyan et al., Cell 81: 505-12 (1995); M. P.Boldin et al., J Biol Chem 270: 7795-8 (1995); F. C. Kischkel etal.,EMBO 14: 5579-5588 (1995)). Briefly, the cytoplasmic domains of DR3(amino acid residues 215-393 (SEQ ID NO:4)) and the death domain mutantADR3 (amino acid residues 215-321 (SEQ ID NO:4) were amplified by PCRusing appropriate templates and primers into pGSTag. pGSTag andpGSTag-TNFR-1 were described previously (A. M. Chinnaiyan et al., Cell81: 505-12 (1995); M. P. Boldin et al., J Biol Chem 270: 7795-8 (1995);F. C. Kischkel et al., EMBO 14: 5579-5588 (1995)). GST and GST fusionproteins were prepared from E. coli strain BL21 (DE3) pLysS usingstandard published procedures and the recombinant proteins immobilizedonto glutathione-agarose beads. ³⁵S-Labeled FADD, RIP and TRADD wereprepared by in vitro transcription-translation using the TNT or T7 orSP6-coupled reticulocyte lysate system from Promega according tomanufacturer's instructions, using pcDNA3 AU1-FADD (A. M. Chinnaiyan etal., Cell 81: 505-12 (1995); M. P. Boldin et al., J Biol Chem 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14: 5579-5588 (1995)), pRKmyc-TRADD (H. Hsu et al., Cell 81: 495-504 (1995)), or pRK myc-RIP (H.Hsu et al., Immunity 4: 387-396 (1996)) as template. Followingtranslation, equal amounts of total ³⁵S-labeled reticulocyte lysate werediluted into 150 μl GST binding buffer (50 mM Tris, pH 7.6,120 mM NaCl,1% NP-40) and incubated for 2 hrs. at 4° C. with the various GST fusionproteins complexed to beads, following the beads were pelleted by pluscentrifugation, washed three times in GST buffer, boiled in SDS-samplebuffer and resolved on a 12.5% SDS-PAGE. Bound proteins were visualizedfollowing autoradioraphy at −80° C. In vitro translated ³⁵S-labeled RIP,TRADD and FADD were incubated with glutathione beads containing GSTalone or GST fusions of the cytoplasmic domain of Fas, TNFR-1, DR3(215-393), or DDR3 (215-321). After the beads were washed, retainedproteins were analyzed by SDS-PAGE and autoradiography. The gel wasCoomassie stained to monitor equivalency of loading.

[0555] To demonstrate the association of DR3 and TRADD in vivo,constructs encoding Flag-TNFR-1 and Flag-ΔTNFR-1 were used. TheFlag-TNFR-1 and Flag-ΔTNFR-1 constructs were described elsewhere (A. M.Chinnaiyan et al., J Biol Chem 271: 4961-4965 (1996)). The constructsencoding Flag-TNFR-1 and Flag-ΔTNFR-1 were described elsewhere (A. M.Chinnaiyan et al., J Biol Chem 271: 4961-4965 (1996)). To facilitateepitope tagging, DR3 and ΔDR3 (1-321) were cloned into the IBI KodakFLAG plasmid (pCMV1FLAG) utilizing the signal peptide provided by thevector. 293 cells (2×10⁶/100 mm plate) were grown in DMEM mediacontaining 10% heat-inactivated fetal bovine serum containing penicillinG, streptomycin, glutamine, and non-essential amino acids. Cells weretransfected using calcium phosphate precipitation with the constructsencoding the indicated proteins in combination with pcDNA3-CrmA (M.Tewari et al., J Biol Chem 270: 3255-60 (1995)) to prevent cell deathand thus maintain protein expression. Cells were lysed in 1 ml lysisbuffer (50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and a proteaseinhibitor cocktail). Lysates were immunoprecipitated with a controlmonoclonal antibody or anti-Flag antibody for at least 4 hrs, at 4° C.as previously described (A. M. Chinnaiyan et al., J Biol Chem 271:4961-4965 (1996)). The beads were washed with lysis buffer 3×, but inthe case of TRADD binding, the NaCl concentration was adjusted to 1M.The precipitates were fractioned on 12.5% SDS-PAGE and transferred tonitrocellulose. Subsequent Western blotting was performed as describedelsewhere (H. Hsu et al., Cell 84: 299-308 (1996); A. M. Chinnaiyan etal., J Biol Chem 271, 4961-4965 (1996)). After 24-32 hours, extractswere prepared and immunoprecipitated with a control monoclonal antibodyor anti-Flag monoclonal antibody (IBI Kodak). Western analysis indicatedthat myc-TRADD and death receptor expression levels were similar in allsamples. Coprecipitating myc-TRADD was detected by immunoblotting usingan anti-myc HRP conjugated antibody (Boehringer Mannheim).

[0556] Results

[0557] As an initial screen, in vitro translated radiolabeled DAMs wereprecipitated with various glutathione S-transferase (GST) fusionproteins immobilized on glutathione-Sepharose beads. As predicted fromprevious studies (A. M. Chinnaiyan et al., Cell 81: 505-12 (1995); M. P.Boldin et al., J Biol Chem 270: 7795-8 (1995); F. C. Kischkel et al.,EMBO 14: 5579-5588 (1995); H. Hsu et al., Cell 81: 495-504 (1995)), FADDassociated with the GST-Fas cytoplasmic domain while TRADD associatedwith the GST-TNFR-1 cytoplasmic domain. In addition, there was a direct,albeit weak, interaction between RIP and GST-TNFR-1. Interestingly,GST-DDCR associated specifically with TRADD, but not FADD or RIP.Furthermore, a truncated death domain mutant of DR3 (GST-DDR3) failed tointeract with TRADD. To demonstrate the association of DR3 and TRADD invivo, 293 cells were transiently transfected with plasmids that directthe synthesis of myc-epitope tagged TRADD (myc-TRADD) and Flag-epitopetagged DR3 (Flag-DR3), Flag-TNFR-1 or mutants. Consistent with the invitro binding study, TRADD specifically coprecipitated with DR3 andTNFR-1, but not with the death domain mutants, DDR3 and DTNFR-1. Thus,it appears that DR3, like TNFR-1, may activate downstream signalingcascades by virtue of its ability to recruit the adapter molecule TRADD.

[0558] Overexpression of TRADD induces apoptosis and NF-kBactivation-two of the most important activities signaled by TNFR-1 (H.Hsu et al., supra). Upon oligomerization of TNFR-1 by trimeric TNF,TRADD is recruited to the receptor-signaling complex (H. Hsu et al.,Cell 84:299-308 (1996)). TRADD can then recruit the following signaltransducing molecules: 1) TRAF2, a TNFR-2- and CD40-associated molecule(M. Rothe et al., Cell 78: 681-92 (1994); M. Rothe et al., Science269:1424-1427 (1995)), that mediates NF-kB activation, 2) RIP,originally identified as a Fas/APO-1-interacting protein by two-hybridanalysis (B. Z. Stanger et al., Cell 81: 513-23 (1995)), that mediatesNF-kB activation and apoptosis (H. Hsu et al., Immunity 4: 387-396(1996)), and 3) FADD, a Fas/APO-1-associated molecule, that mediatesapoptosis (A. M. Chinnaiyan et al., Cell 81: 505-12 (1995); M. P. Boldinet al., J. Biol Chem 270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588 (1995)). Thus, the inventors demonstrate that RIP, TRAF2 andFADD could be co-immunoprecipitated with DR3. In 293 cells expressingDR3 and RIP, only a weak association could be detected between the twomolecules. However, in the presence of TRADD, RIP association with DR3was significantly enhanced. Likewise, very little TRAF2 directlyco-precipitated with DR3 in 293 cells. However, when DR3 and TRAF2 wereexpressed in the presence of TRADD and RIP (both of which can bindTRAF2), an enhanced binding of TRAF2 to DR3 could be detected. A similarassociation between FADD and DR3 was also observed. In the presence ofTRADD, FADD efficiently co-precipitated with DR3.

[0559] Previous studies demonstrated that FADD could recruit theICE/CED-3-like protease FLICE to the Fas/APO-1 death inducing signalingcomplex (M. Muzio et al., Cell 85: 817-827 (1996); M. P. Boldin et al.,Cell 85: 803-815 (1996)). To demonstrate that FLICE can associate withTNFR-1 and DR3, co-precipitation experiments in 293 cells were carriedout. Interestingly, FLICE was found complexed to TNFR-1 and DR3.Co-transfection of TRADD and/or FADD failed to enhance theFLICE-TNFR-1/DR3 interaction, suggesting that endogenous amounts ofthese adapter molecules were sufficient to maintain this association.

EXAMPLE 6

[0560] DR3 Induced Apoptosis and NF-kB Activation

[0561] Overexpression of Fas/APO-1 and TNFR-1 in mammalian cells mimicsreceptor activation (M. Muzio etal., Cell 85: 817-827 (1996); M. P.Boldin etal., Cell 85: 803-815 (1996)). Thus, this system was utilizedto study the functional role of DDCR. Ectopic expression of DR3 in MCF7breast carcinoma cells and 293 human embryonic kidney cells inducedrapid apoptosis.

[0562] Experimental Design

[0563] Cell death assays were performed essentially as previouslydescribed (A. M. Chinnaiyan et al., Cell 81: 505-12 (1995); M. P. Boldinetal., J Biol Chem 270: 7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588 (1995); A. M. Chinnaiyan et al., J Biol Chem 271: 4961-4965(1996)). Briefly, MCF-7 human breast carcinoma clonal cell lines stablytransfected with either vector alone, a CrmA expression construct (M.Tewari et al., J Biol Chem 270:3255-60 (1995)), or FADD-DN expressionconstruct (A. M. Chinnaiyan et al., J Biol Chem 271: 4961-4965 (1996))were transiently transfected with pCMV-β-galatosidase in the presence ofa ten-fold excess of pcDNA3 expression constructs encoding the indicatedproteins using lipofectamine (GIBCO-BRL). 293 cells were likewisetransfected using the CaPO₄ method. The ICE family inhibitor z-VAD-frnk(Enzyme Systems Products, Dublin, Calif.) was added to the cells at aconcentration of 10 μM, 5 hrs after transfection. 32 hours followingtransfection, cells were fixed and stained with X-Gal as previouslydescribed (A. M. Chinnaiyan et al., Cell 81: 505-12 (1995); M. P. Boldinet al., J Biol Chem 270: 7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588 (1995)). The data (mean +/− SD) shown are the percentage ofround blue cells among the total number of blue cells counted. Data wereobtained from at least three independent experiments.

[0564] NF-kB luciferase assays were performed as described elsewhere (H.Hsu et al., Immunity 4: 387-396 (1996); M. D. Adams et al., Nature 377:3-174 (1995); G. S. Feng et al., J Biol Chem 271: 12129-32 (1996); M.Rothe et al., Cell 78: 681-92 (1994); M. Rothe et al., Science269:1424-1427 (1995); A. M. Chinnaiyan et al., J Biol Chem 271:4961-4965 (1996)). Briefly, 293 cells were co-transfected by calciumphosphate precipitation with pCMV-β-galactosidase, E-selectin-luciferasereporter gene (M. Rothe et al., Cell 78: 681-92 (1994); M. Rothe et al.,Science 269:1424-1427 (1995)), the indicated death receptors, and theindicated dominant negative inhibitors. In addition, DR3 or DDR3 wascotransfected with the pLantem expression construct (GIBCO-BRL), whichencodes green fluorescent protein (photographic inset). Cells werevisualized by fluorescence microscopy using a FITC range barrier filtercube. Nuclei of transfected cells were visualized by DAPI staining andthe image overlaid. (Cell death assays were performed essentially aspreviously described (Chinnaiyan et al., Cell 81:505-12 (1995); Boldin,et al., J. Biol. Chem. 270:7795-8 (1995); Kischkel et al., EMBO14:5579-5588 (1995)); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965(1996)). The dominant negative inhibitors were used at a 4-fold higherquantity than the death receptors. Total DNA was kept constant.

[0565] To show that DR3 induces NF-kB activation which may be inhibitedby RIP-DN (Stanger et al., Cell 81:513-23 (1995)) and TRAF2-DN (Hsu etal., Cell 81:495-504 (1995); Rothe et al., Cell 78:681-92 (1994); Rotheet al. Science 269:1424-1427 (1995)), 293 cells were co-transfected withthe indicated molecules and an NF-kB luciferase reporter plasmid (Rotheet al., Cell 78:681-92 (1994); Rothe et al., Science 269:1424-1427(1995)), and luciferase activities subsequently determined. NF-kBluciferase assays were performed as described elsewhere (Hsu et al.,Immunity 4:387-396 (1996); Adams et al., Nature 377:3-174 (1995); Fenget al., J. Biol. Chem. 271:12129-32 (1996); Rothe et al., Cell 78:681-92(1994); Rothe et al. Science 269:1424-1427 (1995); Chinnaiyan et al., J.Biol. Chem. 271:4961-4965 (1996)). Briefly, 293 cells wereco-transfected by calcium phosphate precipitation withpCMB-β-galactosidase, E-selectin-luciferase reporter gene (Rothe et al.,Cell 78:681-92 (1994); Rothe et al., Science 269:1424-1427 (1995)), theindicated death receptors, and the indicated dominant negativeinhibitors. The dominant negative inhibitors were used at a 4-foldhigher quantity than the death receptors. Total DNA was kept constant.Representative experiment performed in duplicate three independent times(mean ± SD).

[0566] Results

[0567] The cells displayed morphological alterations typical of cellsundergoing apoptosis, becoming rounded, condensed and detaching from thedish. In MCF7 cells, plasmids encoding full-length DR3 or DDR3 wereco-transfected with the pLantern reporter construct encoding greenfluorescent protein. Nuclei of cells transfected with DR3, but not DDR3,exhibited apoptotic morphology as assessed by DAPI staining. Similar toTNFR-1 and Fas/APO-1 (M. Muzio et al., Cell 85: 817-827 (1996); M. P.Boldin et al., Cell 85: 803-815 (1996); M. Tewari et al., J Biol Chem270: 3255-60 (1995)), DR3-induced apoptosis was blocked by theinhibitors of ICE-like proteases, CrmA and z-VAD-fmk. Importantly,apoptosis induced by DR3 was also blocked by dominant negative versionsof FADD (FADD-DN) or FLICE (FLICE-DN/MACHa1C360S), which were previouslyshown to inhibit death signaling by Fas/APO-1 and TNFR-1 (M. Muzio etal., Cell 85: 817-827 (1996); M. P. Boldin et al., Cell 85: 803-815(1996); H. Hsu et al., Cell 84: 299-398 (1996); A. M. Chinnaiyan et al.,J Biol Chem 271: 4961-4965 (1996)). Thus, FADD and the ICE-like proteaseFLICE are likely necessary components of DR3-induced apoptosis.

[0568] As DR3 activation recruits three molecules implicated inTNF-induced NF-kB activation, we examined whether DR3 could activateNF-kB. Transfection of a control vector or expression of Fas/APO-1failed to induce NF-kB activation. By contrast, NF-kB was activated byectopic expression of DR3 or TNFR-1, but not by the inactive signalingmutants DDR3 or DTNFR-1. Importantly, DR3-induced NF-kB activation wasblocked by dominant negative derivatives of RIP (RIP-DN) and TRAF2(TRAF2-DN), which were previously shown to abrogate TNF-induced NF-kBactivation (H. Hsu et al., Cell 84: 299-398 (1996); H. Hsu et al.,Immunity 4: 387-396 (1996)). As expected, FADD-DN did not interfere withDR3-mediated NF-kB activation (H. Hsu et al., Cell 84: 299-398 (1996);A. M. Chinnaiyan et al., J Biol Chem 271: 4961-4965 (1996)).

[0569] Thus, the experiments set forth in Examples 6 and 7 demonstratethat DR3 is a death domain-containing molecule capable of triggeringboth apoptosis and NF-kB activation, two pathways dominant in theregulation of the immune system. The experiments also demonstrate theinternal signal transduction machinery of this novel cell deathreceptor. The DR3 signaling complex assembles in a hierarchical mannerwith the recruitment of the multivalent adapter molecule TRADD, fromwhich two distinct signaling cascades emanate: 1) NF-kB activationmediated by TRAF2 and RIP and 2) cell death mediated by FADD, FLICE, andRIP.

EXAMPLE 7

[0570] Gene Therapy Using Endogenous DR3 Gene

[0571] Another method of gene therapy according to the present inventioninvolves operably associating the endogenous DR3 sequence with apromotervia homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411, published Sep. 26, 1996; International PublicationNumber WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989). This method involves the activation of a gene whichis present in the target cells, but which is not expressed in the cells,or is expressed at a lower level than desired. Polynucleotide constructsare made which contain a promoter and targeting sequences, which arehomologous to the 5′ non-coding sequence of endogenous DR3, flanking thepromoter. The targeting sequence will be sufficiently near the 5′ end ofDR3 so the promoter will be operably linked to the endogenous sequenceupon homologous recombination. The promoter and the targeting sequencescan be amplified using PCR. Preferably, the amplified promoter containsdistinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the3′ end of the first targeting sequence contains the same restrictionenzyme site as the 5′ end of the amplified promoter and the 5′ end ofthe second targeting sequence contains the same restriction site as the3′ end of the amplified promoter.

[0572] The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

[0573] In this Example, the polynucleotide constructs are administeredas naked polynucleotides via electroporation. However, thepolynucleotide constructs may also be administered withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, precipitating agents, etc. Such methods of delivery areknown in the art.

[0574] Once the cells are transfected, homologous recombination willtake place resulting in the promoter being operably linked to theendogenous DR3 sequence. This results in the expression of DR3-V 1 orDR3 in the cell. Expression may be detected by immunological staining orany other method known in the art.

[0575] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in DMEM+10% fetal calf serum. Exponentiallygrowing or early stationary phase fibroblasts are trypsinized and rinsedfrom the plastic surface with nutrient medium. An aliquot of the cellsuspension is removed for counting, and the remaining cells aresubjected to centrifugation. The supernatant is aspirated and the pelletis resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3,137 mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

[0576] Plasmid DNA is prepared according to standard techniques. Forexample, to construct a plasmid for targeting to the DR3 locus, plasmidpUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamIsite on the 3′ end. Two DR3 non-coding sequences are amplified via PCR:one DR3 non-coding sequence (DR3 fragment 1) is amplified with a HindIIIsite at the 5′ end and an XbaI site at the 3′ end; the other DR3non-coding sequence (DR3 fragment 2) is amplified with a BamHI site atthe 5′ end and a HindIII site at the 3′ end. The CMV promoter and DR3fragments are digested with the appropriate enzymes (CMV promoter—XbaIand BamHI; DR3 fragment 1—XbaI; DR3 fragment 2—BamHI) and ligatedtogether. The resulting ligation product is digested with HindIII, andligated with the HindIII-digested pUC18 plasmid.

[0577] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrodegap (Bio-Rad). The final DNA concentration is generally at least 120μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5×10⁶cells) is then added to the cuvette, and the cell suspension and DNAsolutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

[0578] Electroporated cells are maintained at room temperature forapproximately 5 minutes, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

[0579] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product. Thefibroblasts can then be introduced into a patient as described above.

EXAMPLE 8

[0580] Production of an Antibody

[0581] Hybridoma Technology

[0582] The antibodies of the present invention can be prepared by avariety of methods. (See, Ausubel et al., eds., 1998, Current Protocolsin Molecular Biology, John Wiley & Sons, New York, Chapter 2.) As oneexample of such methods, cells expressing DR3-V1 or DR3 are administeredto an animal to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of DR3-V1 or DR3protein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

[0583] Monoclonal antibodies specific for protein DR3-V1 or DR3 areprepared using hybridoma technology. (Kohler et al., Nature 256:495(1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.,Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)).In general, an animal (preferably a mouse) is immunized with DR3-V1 orDR3 polypeptide or, more preferably, with a secreted DR3-V1 or DR3polypeptide-expressing cell. Such polypeptide-expressing cells arecultured in any suitable tissue culture medium, preferably in Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin.

[0584] The splenocytes of such mice are extracted and fused with asuitable myeloma cell line. Any suitable myeloma cell line may beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP20), available fromthe ATCC. After fusion, the resulting hybridoma cells are selectivelymaintained in HAT medium, and then cloned by limiting dilution asdescribed by Wands et al. (Gastroenterology 80:225-232 (1981). Thehybridoma cells obtained through such a selection are then assayed toidentify clones, which secrete antibodies capable of binding the DR3-V1or DR3 polypeptide.

[0585] Alternatively, additional antibodies capable of binding to DR3-V1or DR3 polypeptide can be produced in a two-step procedure usinganti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and therefore, it is possible toobtain an antibody, which binds to a second antibody. In accordance withthis method, protein specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theDR3-V1 or DR3 protein-specific antibody can be blocked by DR3-V1 or DR3.Such antibodies comprise anti-idiotypic antibodies to the DR3-V 1 or DR3protein-specific antibody and are used to immunize an animal to induceformation of further DR3-V1 or DR3 protein-specific antibodies.

[0586] For in vivo use of antibodies in humans, an antibody is“humanized”. Such antibodies can be produced using genetic constructsderived from hybridoma cells producing the monoclonal antibodiesdescribed above. Methods for producing chimeric and humanized antibodiesare known in the art and are discussed infra. (See, for review,Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985)).

[0587] Isolation of Antibody Fragments Directed Against DR3-V1 and DR3From a Library of scFvs

[0588] Naturally occurring V-genes isolated from human PBLs areconstructed into a large library of antibody fragments which containreactivities against polypeptides of the present invention to which thedonor may or may not have been exposed (see, e.g., U.S. Pat. No.5,885,793 incorporated herein in its entirety by reference).

[0589] Rescue of the Library

[0590] A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harboring the phagemid are used to inoculate50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin(2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml of thisculture is used to innoculate 50 ml of 2×TY-AMP-GLU, 2×10⁸ TU of deltagene 3 helper phage (M13 delta gene III, see WO92/01047) are added andthe culture incubated at 37° C. for 45 minutes without shaking and thenat 37° C. for 45 minutes with shaking. The culture is centrifuged at4000 r.p.m. for 10 minutes and the pellet resuspended in 2 liters of2×TY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grownovernight. Phages are prepared as described in WO92/01047.

[0591] M13 delta gene III is prepared as follows: M13 delta gene IIIhelper phage does not encode gene III protein, hence the phage(mid)sdisplaying antibody fragments have a greater avidity of binding toantigen. Infectious M13 delta gene III particles are made by growing thehelper phage in cells harboring a pUC19 derivative supplying the wildtype gene III protein during phage morphogenesis. The culture isincubated for 1 hour at 37° C. without shaking and then for a furtherhour at 37° C. with shaking. Cells are pelleted (IEC-Centra 8, 4000revs/min for 10 min), resuspended in 300 ml 2×TY broth containing 100 μgampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight,shaking at 37° C. Phage particles are purified and concentrated from theculture medium by two PEG-precipitations (Sambrook et al., 1990)resuspended in 2 ml PBS and passed through a 0.45 μm filter (MinisartNML; Sartorius) to give a final concentration of approximately 10¹³transducing units/ml (ampicillin-resistant clones).

[0592] Panning of the Library

[0593] Immunotubes (Nunc) are coated overnight in PBS with 4 ml ofeither 100 mg/ml or 10 mg/ml of a polypeptide of the present invention.Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and thenwashed 3 times in PBS. Approximately 10¹³ TU of phage are applied to thetube and incubated for 30 minutes at room temperature tumbling on anover and under turntable and then left to stand for another 1.5 hours.Tubes are washed 10 times with PBS, 0.1% Tween-20 and 10 times with PBS.Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15minutes on an under and over turntable after which the solution isimmediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phages arethen used to infect 10 ml of mid-log E. coli TG1 by incubating elutedphage with bacteria for 30 minutes at 37° C. The E. coli are then platedon TYE plates containing 1% glucose and 100 μg/ml ampicillin. Theresulting bacterial library is then rescued with delta gene 3 helperphage as described above to prepare phage for a subsequent round ofselection. This process is then repeated for a total of 4 rounds ofaffinity purification with tube-washing increased to 20 times with PBS,0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

[0594] Characterization of Binders

[0595] Eluted phage from the 3rd and 4th rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks et al., J.Mol. Biol. 222:581-597 (1991)) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., WO92/01047) and then by sequencing.

EXAMPLE 9

[0596] Method of Determining Alterations in the DR3 Gene

[0597] RNA is isolated from entire families or individual patientspresenting with a phenotype of interest (such as a disease). cDNA isthen generated from these RNA samples using protocols known in the art.(See, Sambrook et al., 1990) The cDNA is then used as a template forPCR, employing primers surrounding regions of interest in SEQ ID NO:1.Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds;60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffersolutions described in Sidransky, D., et al., Science 252:706 (1991).

[0598] PCR products are then sequenced using primers labeled at their 5′end with T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofDR3 are also determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations in DR3 are thencloned and sequenced to validate the results of the direct sequencing.

[0599] PCR products of DR3 are cloned into T-tailed vectors as describedin Holton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156(1991) and sequenced with T7 polymerase (United States Biochemical).Affected individuals are identified by mutations in DR3 not present inunaffected individuals.

[0600] Genomic rearrangements are also observed as a method ofdetermining alterations in the DR3 gene. Genomic clones isolated usingtechniques known in the art are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson, C. et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to theDR3 genomic locus.

[0601] Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, C. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of DR3 (hybridized by the probe) areidentified as insertions, deletions, and translocations. These DR3alterations are used as a diagnostic marker for an associated disease.

EXAMPLE 10

[0602] Method of Detecting Abnormal Levels of DR3 in a Biological Sample

[0603] DR3 polypeptides can be detected in a biological sample, and ifan increased or decreased level of DR3 is detected, this polypeptide isa marker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

[0604] For example, antibody-sandwich ELISAs are used to detect DR3 in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to DR3, at a final concentration of 0.2to 10 μg/ml. The antibodies are either monoclonal or polyclonal and areproduced using technique known in the art. The wells are blocked so thatnon-specific binding of DR3 to the well is reduced.

[0605] The coated wells are then incubated for >2 hours at RT with asample containing DR3. Preferably, serial dilutions of the sample shouldbe used to validate results. The plates are then washed three times withdeionized or distilled water to remove unbounded DR3.

[0606] Next, 50 μl of specific antibody-alkaline phosphatase conjugate,at a concentration of 25-400 ng, is added and incubated for 2 hours atroom temperature. The plates are again washed three times with deionizedor distilled water to remove unbounded conjugate.

[0607] 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution is then added to each well andincubated 1 hour at room temperature to allow cleavage of the substrateand fluorescence. Fluorescence is measured using a microtiter platereader. A standard curve is prepared using the experimental results fromserial dilutions of a control sample with the sample concentrationplotted on the X-axis (log scale) and fluorescence or absorbance on theY-axis (linear scale). The DR3 polypeptide concentration in a sample isthen interpolated using the standard curve based on the measuredfluorescence of that sample.

EXAMPLE 11

[0608] Method of Treating Increased Levels of DR3

[0609] The present invention relates to a method for treating anindividual in need of a decreased level of DR3 biological activity inthe body comprising, administering to such an individual a compositioncomprising a therapeutically effective amount of DR3 antagonist.Preferred antagonists for use in the present invention are DR3-specificantibodies.

[0610] Moreover, it will be appreciated that conditions caused by adecrease in the standard or normal expression level of DR3 in anindividual can be treated by administering DR3, preferably in a solubleand/or secreted form. Thus, the invention also provides a method oftreatment of an individual in need of an increased level of DR3polypeptide comprising administering to such an individual apharmaceutical composition comprising an amount of DR3 to increase thebiological activity level of DR3 in such an individual.

[0611] For example, a patient with decreased levels of DR3 polypeptidereceives a daily dose 0.1-100 μg/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in a soluble and/orsecreted form.

EXAMPLE 12

[0612] Method of Treating Decreased Levels of DR3

[0613] The present invention also relates to a method for treating anindividual in need of an increased level of DR3 biological activity inthe body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of DR3 or an agonistthereof.

[0614] Antisense technology is used to inhibit production of DR3. Thistechnology is one example of a method of decreasing levels of DR3polypeptide, preferably a soluble and/or secreted form, due to a varietyof etiologies, such as cancer.

[0615] For example, a patient diagnosed with abnormally increased levelsof DR3 is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if it is determined to be well tolerated.

EXAMPLE 13

[0616] Method of Treatment Using Gene Therapy—Ex Vivo

[0617] One method of gene therapy transplants fibroblasts, which arecapable of expressing soluble and/or mature DR3 polypeptides, onto apatient. Generally, fibroblasts are obtained from a subject by skinbiopsy. The resulting tissue is placed in tissue-culture medium andseparated into small pieces. Small chunks of the tissue are placed on awet surface of a tissue culture flask approximately ten pieces beingplaced in each flask. The flask is turned upside down, closed tight andleft at room temperature over night. After 24 hours at room temperature,the flask is inverted and the chunks of tissue remain fixed to thebottom of the flask and fresh media (e.g., Ham's F12 media, with 10%FBS, penicillin and streptomycin) is added. The flasks are thenincubated at 37° C. for approximately one week.

[0618] At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

[0619] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flankedby the long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0620] The cDNA encoding DR3 can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end encoding sequences respectively.Preferably, the 5′ primer contains an EcoRI site and the 3′ primerincludes a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the amplified EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is then used to transform E. coli HB101,which are then plated onto agar containing kanamycin for the purpose ofconfirming that the vector contains properly inserted DR3.

[0621] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the DR3 gene is then added to the media and thepackaging cells transduced with the vector. The packaging cells nowproduce infectious viral particles containing the DR3 gene (thepackaging cells are now referred to as producer cells).

[0622] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a Millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his. Once thefibroblasts have been efficiently infected, the fibroblasts are analyzedto determine whether DR3 protein is produced.

[0623] The engineered fibroblasts are then transplanted onto the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads.

EXAMPLE 14

[0624] Method of Treatment Using Gene Therapy—In Vivo

[0625] Another aspect of the present invention is using in vivo genetherapy methods to treat disorders, diseases and conditions. The genetherapy method relates to the introduction of naked nucleic acid (DNA,RNA, and antisense DNA or RNA) DR3 sequences into an animal to increaseor decrease the expression of the DR3 polypeptide. The DR3polynucleotide may be operatively linked to a promoter or any othergenetic elements necessary for the expression of the DR3 polypeptide bythe target tissue. Such gene therapy and delivery techniques and methodsare known in the art, see, for example, WO90/11092, WO98/11779; U.S.Pat. No. 5,693,622, 5,705,151, 5,580,859; Tabata H. et al., Cardiovasc.Res. 35:470-479 (1997); Chao J. et al., Pharmacol. Res. 35:517-522(1997); Wolff J. A. Neuromuscul. Disord. 7:314-318 (1997); Schwartz B.et al,. Gene Ther. 3:405-411 (1996); Tsurumi Y. et al., Circulation94:3281-3290 (1996) (incorporated herein by reference).

[0626] The DR3 polynucleotide constructs may be delivered by any methodthat delivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The DR3 polynucleotide constructscan be delivered in a pharmaceutically acceptable liquid or aqueouscarrier.

[0627] The term “naked” polynucleotide, DNA or RNA, refers to sequencesthat are free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the DR3 polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner, P. et al. Ann.NY Acad. Sci. 772:126-139 (1995), and Abdallah, B. et al. Biol. Cell85:1-7 (1995)) which can be prepared by methods well known to thoseskilled in the art.

[0628] The DR3 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapy techniques, onemajor advantage of introducing naked nucleic acid sequences into targetcells is the transitory nature of the polynucleotide synthesis in thecells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

[0629] The DR3 polynucleotide construct can be delivered to theinterstitial space of tissues within an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

[0630] For the naked DR3 polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 μg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DR3polynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

[0631] The dose response effects of injected DR3 polynucleotide inmuscle in vivo are determined as follows. Suitable DR3 template DNA forproduction of mRNA coding for DR3 polypeptide is prepared in accordancewith a standard recombinant DNA methodology. The template DNA, which maybe either circular or linear, is either used as naked DNA or complexedwith liposomes. The quadriceps muscles of mice are then injected withvarious amounts of the template DNA.

[0632] Five to six week old female and male Balb/C mice are anesthetizedby intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cmincision is made on the anterior thigh, and the quadriceps muscle isdirectly visualized. The DR3 template DNA is injected in 0.1 ml ofcarrier in a 1 cc syringe through a 27 gauge needle over one minute,approximately 0.5 cm from the distal insertion site of the muscle intothe knee and about 0.2 cm deep. A suture is placed over the injectionsite for future localization, and the skin is closed with stainlesssteel clips.

[0633] After an appropriate incubation time (e.g., 7 days) muscleextracts are prepared by excising the entire quadriceps. Every fifth 15μm cross-section of the individual quadriceps muscles is histochemicallystained for DR3 protein expression. A time course for DR3 proteinexpression may be done in a similar fashion except that, quadriceps fromdifferent mice are harvested at different times. Persistence of DR3 DNAin muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using DR3 naked DNA.

EXAMPLE 15

[0634] TNFR6-alpha and DR3 Interact with TNF-Gamma-beta

[0635] The premyeloid cell line TF-1 was stably transfected withSRE/SEAP (Signal Response Element/Secreted Alkaline Phosphatase)reporter plasmid that responds to the SRE signal transduction pathway.The TF1/SRE reporter cells were treated with TNF-gamma-beta(International Publication Numbers WO96/14328, WO00/66608, andWO00/08139) at 200 ng/ml and showed activation response as recorded bythe SEAP activity. This activity can be neutralized by A TNFR6-alpha Fcfusion protein (hereinafter TR6.Fc in this example) in a dose dependentmanner. The TR6.Fc by itself, in contrast, showed no activity on theTF1/SRE reporter cells. The results demonstrate that 1) TF-1 is a targetcell for TNF-gamma-beta ligand activity, and 2) TR6 interacts withTNF-gamma-beta and inhibits its activity on TF-1 cells.

[0636] Similarly, the interaction of DR3 (International PublicationNumbers WO97/33904 and WO/0064465) and TNF-gamma-beta can bedemonstrated using TF-1/SRE reporter cells. The results indicate thatDR3.Fc interacts with TNF-gamma-beta, either by competing naturallyexpressed DR3 on TF-1 cells or forming inactive TNF-gamma-beta /DR3.fccomplex, or both. At least three additional pieces of evidencedemonstrate an interaction between TNF-gamma-beta and DR3 and TR6.First, both TR6.Fc and DR3.Fc are able to inhibit TNF-gamma-betaactivation of NF-κB in 293T cells, whereas in the same experiment,TNFRI.Fc was not able to inhibit TNF-gamma-beta activation of NF-κB in293T cells. Secondly, both TR6.Fc and DR3.Fc can be used toimmunoprecipitate TNF-gamma-beta. Thirdly, TR6.Fc proteins can bedetected by FACS analysis to specifically bind cells transfected withTNF-gamma-beta.

EXAMPLE 16

[0637] TNF-gamma-beta is a Novel Ligand for DR3 and TR6-alpha (DcR3) andFunctions as a T cell Costimulator

[0638] Introduction

[0639] Members of the TNF and TNFR superfamilies of proteins areinvolved in the regulation of many important biological processes,including development, organogenesis, innate and adaptive immunity(Locksley et al., Cell 104:487-501 (2001)). Interaction of TNF ligandssuch as TNF, Fas, LIGHT and BLyS with their cognate receptor (orreceptors) has been shown to affect the immune responses, as they areable to activate signaling pathways that link them to the regulation ofinflammation, apoptosis, homeostasis, host defense, and autoimmunity.The TNFR superfamily can be divided into two groups based on thepresence of different domains in the intracellular portion of thereceptor. One group contains a TRAF binding domain that enables them tocouple to TRAFs (TNFR-associated factor); these in turn activate asignaling cascade that results in the activation of NF-κB and initiationof transcription. The other group of receptors is characterized by a 60amino acid globular structure named Death Domain (DD). Historicallydeath domain-containing receptors have been described as inducers ofapoptosis via the activation of caspases. These receptors include TNFR1,DR3, DR4, DR5, DR6 and Fas. More recent evidence (Siegel et al., NatureImmunology 1:469-474 (2000) and references within) has shown that somemembers of this subgroup of receptors, such as Fas, also have theability to positively affect T cell activation. A third group ofreceptors has also been described. The members of this group, thatinclude DcR1, DcR2, OPG, and TNFR-6 alpha (also called DcR3, andhereinafter in this example referred to as “TR6”), have been named decoyreceptors, as they lack a cytoplasmic domain and may act as inhibitorsby competing with the signal transducing receptor for the ligand(Ashkenazi etal., Curr. Opin. Cell Biol. 11:255-260(1999)). TR6, whichexhibits closest homology to OPG, associates with high affinity to FasLand LIGHT, and inhibits FasL-induced apoptosis both in vitro and in vivo(Pitti et al., Nature 396:699-703 (1998), Yu et al., J. Biol. Chem.274:13733-6 (1999); Connolly et al., J. Pharmacol. Exp. Ther. 298:25-33(2001)). Its role in down-regulating immune responses was stronglysuggested by the observation that TR6 supresses T-cell responses againstalloantigen (Zhang et al., J. Clin. Invest. 107:1459-68 (2001)) andcertain tumors overexpressTR6 (Pitti etal., supra; Bai et al., Proc.Natl. Acad. Sci. 97:1230-1235 (2000)).

[0640] DR3 is a DD-containing receptor that shows highest homology toTNFR1 (Chinnaiyan et al., Science 274:990-2 (1996); Kitson et al.,Nature 384:372-5 (1996); Marsters et al., Curr. Biol. 6:1669-76 (1996);Bodmer et al., Immunity 6:79-88 (1997); Screaton et al., Proc. Natl.Acad. Sci. 94:4615-19 (1997); Tan et al., Gene 204:35-46 (1997)). Incontrast to TNFR1, which is ubiquitously expressed, DR3 appears to bemostly expressed by lymphocytes and is efficiently induced following Tcell activation. TWEAK/Apo3L was previously shown to bind DR3 in vitro(Marsters et al., Curr. Biol. 8:525-528 (1998)). However, more recentwork raised doubt about this interaction and showed that TWEAK was ableto induce NF-κB and caspase activation in cells lacking DR3 (Schneideret al., Eur. J. Immunol. 29:1785-92 (1999); Kaptein et al., FEBS Letters485:135-141 (2000)).

[0641] In this Example, the characterization of the ligand,TNF-gamma-beta (also known as TL1β; described in InternationalPublication Numbers: WO00/08139 and WO00/66608 which are hereinincorporated by reference in their entireties), for both DR3 andTR6/DcR3 is described. TNF-gamma-beta is a longer variant ofTNF-gamma-alpha (also known as VEGI and TL1; described in InternationalPublication Numbers WO96/14328, WO99/23105, WO00/08139 and WO00/66608which are herein incorporated by reference in their entireties), whichwas previously identified as an endothelial-derived factor thatinhibited endothelial cell growth in vitro and tumor progression in vivo(Tan et al., Gene 204:35-46 (1997); Zhai et al., FASEB J. 13:181-9(1999); Zhai et al., Int. J. Cancer 82:131-6 (1999); Yueetal., J. Biol.Chem. 274:1479-86 (1999)). It was found that TNF-gamma-beta is the moreabundant form than TNF-gamma-alpha and is upregulated by TNFα and IL-1α.U.S. Pat. No. 5,876,969.

[0642] As shown herein, the interaction between TNF-gamma-beta and DR3in 293T cells and in the erythroleukemic line TF-1 results in activationof NF-κB and induction of caspase activity, respectively. TR6 is able toinhibit these activities by competing with DR3 for TNF-gamma-beta. Moreimportantly, it was found that in vitro, TNF-gamma-beta functionsspecifically on activated T cells to promote survival and secretion ofthe proinflammatory cytokines IFNγ and GMCSF, and it markedly enhancesacute graft-versus-host reactions in mice.

[0643] Results

[0644] TNF-gamma-beta is a Longer Variant of TNF-gamma-alpha, a Memberof the TNF Superfamily of Ligands

[0645] To identify novel TNF like molecules, a database of over threemillion human expressed sequence tag (EST) sequences was analyzed usingthe BLAST algorithm. Several EST clones with high homology to TNF likemolecule 1, TNF-gamma-alpha (Tan et al., Gene 204:35-46 (1997); Zhai etal., FASEB J. 13:181-9 (1999); Yue et al., J. Biol. Chem 274:1479-86(1999)) were identified from endothelial cell CDNA libraries. Sequenceanalysis of these cDNA clones revealed a 2080 base pair (bp) insertencoding an open reading frame of 251 amino acids (aa) with two upstreamin-frame stop codons. The predicted protein lacks a leader sequence butcontains a hydrophobic transmembrane domain near the N-terminus, and acarboxyl domain that shares 20-30% sequence similarity with other TNFfamily members. Interestingly, the C-terminal 151-aa of this protein(residues 101-251) is identical to residues 24 to 174 ofTNF-gamma-alpha, whereas the amino-terminal region shares no sequencesimilarity. The predicted extracellular receptor-interaction domain ofTNF-gamma-beta contains two potential N-linked glycosylation sites andshows highest amino acid sequence identity to TNF (24.6%), followed byFasL (22.9%) and LTα (22.2%). A 337-bp stretch of the TNF-gamma-betacDNA, containing most of the 5′ untranslated region and the sequencesencoding the first 70 amino acids of the TNF-gamma-beta protein, matchesa genomic clone on human chromosome 9 (Genbank Accession: AL390240,clone RP11-428F18). Further analysis of the human genomic sequencesreveals that TNF-gamma-alpha and TNF-gamma-beta are likely derived fromthe same gene. While TNF-gamma-beta is encoded by four putative exons,similar to most TNF-like molecules, TNF-gamma-alpha is encoded by onlythe last exon and the extended N-terminal intron region, and thereforelacks a putative transmembrane domain and the first conserved β-sheet

[0646] Mouse and rat TNF-gamma-beta cDNAs isolated from normal kidneycDNAs each encode a 252-aa protein. The overall amino acid sequencehomology between human and mouse, and human and rat TNF-gamma-betaproteins is 63.7% and 66.1%, respectively. Higher sequence homology wasfound in the predicted extracellular receptor-interaction domains, ofwhich human and mouse share 71.8% and human and rat share 75.1% sequenceidentity. An 84.2% sequence identity is seen between the mouse and ratTNF-gamma-beta proteins.

[0647] Like most TNF ligands, TNF-gamma-beta exists as a membrane-boundprotein and can also be processed into a soluble form when ectopicallyexpressed. The N-terminal sequence of soluble TNF-gamma-beta proteinpurified from full-length TNF-gamma-beta transfected 293T cells wasdetermined to be Leu 72.

[0648] TNF-gamma-beta is Predominantly Expressed by Endothelial Cells, aMore Abundant Form Than TNF-gamma-alpha, and is Inducible by TNF andIL-1α

[0649] To determine the expression pattern of TNF-gamma-beta,TNF-gamma-beta specific primer and fluorescent probe were used forquantitative real-time polymerase chain reaction (TaqMan) and reversetranscriptase polymerase chain reaction (RT-PCR) (see ExperimentalProcedures below). TNF-gamma-beta is expressed predominantly by humanendothelial cells, including umbilical vein endothelial cells (HUVEC),adult dermal microvascular endothelial cells (HMVEC-Ad) and uterusmyometrial endothelial cells (UtMEC-Myo), with highest expression seenin HUVEC. A ˜750 bp DNA fragment was readily amplified from theseendothelial cells by RT-PCR, indicating the presence of full-lengthTNF-gamma-beta transcripts. Very little expression was seen in humanaortic endothelial cells (HAEC) or other human primary cells includingadult dermal fibroblast (NHDF-Ad and HFL-1), aortic smooth muscle cells(AoSMC), skeletal muscle cells (SkMC), adult keratinocytes (NHEK-Ad),tonsillar B cells, T cells, NK cells, monocytes, or dendritic cells.Consistent with these results, TNF-gamma-beta RNA was detected in humankidney, prostate, stomach, and low levels were seen in intestine, lung,and thymus, but not in heart, brain, liver, spleen, or adrenal gland. Nosignificant levels of TNF-gamma-beta mRNA in any of the cancer celllines tested, including 293T, HeLa, Jurkat, Molt4, Raji, IM9, U937,Caco-2, SK-N-MC, HepG2, KS4-1, and GH4C were detected.

[0650] As the expression pattern of TNF-gamma-beta is very similar tothat of TNF-gamma-alpha (Tan et al., Gene 204:35-46 (1997); Zhai et al.,FASEB J. 13:181-9 (1999)), the relative abundance of the two RNA specieswas analyzed using TNF-gamma-alpha and TNF-gamma-beta specific primersand fluorescence probes for conventional and quantitative RT-PCR. MoreTNF-gamma-beta mRNA was detected than that of TNF-gamma-alpha using bothmethods. The amount of TNF-gamma-beta mRNA is at least 15-fold higherthan that of TNF-gamma-alpha in the same RNA samples. To determine ifTNF-gamma-beta mRNA levels were inducible HUVEC cells were stimulatedwith either TNF, IL-1α, PMA, bFGF or IFNγ. PMA and IL-1α rapidly inducedhigh levels of TNF-gamma-beta mRNA, with a peak in expression reached at6 hours after treatment. TNF was also able to induce TNF-gamma-betamRNA, but the time course of induction appeared to be delayed comparedto PMA and IL-1α. In contrast, bFGF and IFNγ did not significantlyaffect the expression of TNF-gamma-beta. TNF-gamma-beta protein levelsin the supernatants of activated HUVEC cells were analyzed by ELISA anda similar profile of induction was observed.

[0651] Identification of DR3 and TR6 as Receptors for TL1β

[0652] To identify the receptor for TNF-gamma-beta, we generated HEK293Fstable transfectants expressing full length TNF-gamma-beta on the cellsurface (confirmed by Taqman and flow cytometric analysis usingTNF-gamma-beta monoclonal antibody). These cells were used to screen theFc-fusion form of the extracellular domain of TNFR family members,including TNFR1, Fas, HveA, DR3, DR4, DR5, DR6, DcR1, DcR2, TR6, OPG,RANK, AITR, TACI, CD40, and OX40. DR3-Fc and TR6-Fc bound efficiently tocells expressing TNF-gamma-beta but not to vector control transfectedcells. In contrast, HveA-Fc and all the other receptors tested did notbind to the TNF-gamma-beta expressing cells. TR6 has been previouslydescribed as a decoy receptor (Pitti et al., Nature 396:699-703 (1998);Yu et al., J. Biol Chem. 274:13733-6 (1999)) capable of competing withFas and HveA for binding of FasL and LIGHT, respectively. Whether TR6could compete with DR3 for TNF-gamma-beta binding was tested. When a 2:1molar ratio of a non-tagged form of TR6 and DR3-Fc were used, no bindingof DR3-Fc was detected on TNF-gamma-beta expressing cells. These resultsdemonstrated that both DR3 and TR6 can bind to membrane-bound form ofthe TNF-gamma-beta protein.

[0653] Whether TNF-gamma-beta protein could bind to membrane-bound formof the receptor, DR3 was tested. A FLAG-tagged soluble form of the TL1β(aa 72-251) protein was tested for binding of cells transientlytransfected with different members of the TNFR family, including TNFR2,LTβ R,4-1BB, CD27, CD30, BCMA, DR3, DR4, DR5, DR6, DcR1, DcR2, RANK,HveA, and AITR. Binding of FLAG-TL1β to cells expressing full length orDD-deleted DR3, but not to any of the other receptors tested, wasconsistently detected, demonstrating that TNF-gamma-beta interacts withmembrane-associated DR3. The small shift (˜30%) seen when full lengthDR3 was used is likely due to the presence of low DR3-expressing cellswhile DR3 overexpressed cells undergone apoptosis.

[0654] Coimmunoprecipitation studies were also performed to confirm thatTNF-gamma-beta could specifically bind DR3 and TR6. Consistent with whatwe observed in FACS analysis, we found that DR3-Fc and TR6-Fcspecifically interacted with FLAG-TNF-gamma-beta. In contrast, Fas-Fc orTACI-Fc could not immunoprecipitate FLAG-TNF-gamma-beta, but efficientlybound their known ligands, FLAG-FasL and FLAG-BlyS, respectively.

[0655] To verify that the TNF-gamma-beta binding to DR3 and TR6 wasspecific and exhibited characteristics that were similar to thoseobserved with other TNF family members to their cognate receptors, aBIAcore analysis using a non-tagged TNF-gamma-beta (aa 72-251) proteinpurified from E. coli was performed. The kinetics of TNF-gamma-betabinding to DR3-Fc was determined using three different batches of theTNF-gamma-beta protein. The ka and kd values were found to be 6.39E+05Ms⁻¹ and 4.13E-03M⁻¹, respectively. The average Kd value was 6.45±0.2nM. TNF-gamma-beta was also examined for its ability to bind to severalother TNF-related receptors (HveA, BCMA, TACI, and TR6). In addition toDR3, only TR6 was found to have significant and specific binding toTNF-gamma-beta. The ka and kd values were 1.04E+06 Ms⁻¹ and 1.9E−03 M⁻,respectively, which gives a Kd of 1.8 nM. The specificity of binding ofTL1β to DR3-Fc and TR6-Fc were confirmed by the competition ofTNF-gamma-beta binding in the presence of excess soluble receptor-Fc.These Kd values for binding of TNF-gamma-beta to DR3-Fc and TR6-Fc arecomparable to those determined for other TNFR-ligand interactions.

[0656] Interaction of TL1β with DR3 Induces Activation of NF-κB

[0657] Previous reports have demonstrated that ectopic expression of DR3results in the activation of the transcription factor NF-κB (Chinnaiyanet al., Science 274:990-2 (1996); Kitson et al., Nature 384:372-5(1996), Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmer et al.,Immunity 6:79-88 (1,997)). TNF-gamma-beta induced signaling in areconstituted system in 293T cells, into which DR3 and a NF-κB-SEAPreporter had been introduced by transient transfection, was studied. Toavoid spontaneous apoptosis or NF-κB activation accompanied with DR3overexpression, a limited amount of DR3-expression DNA, that by itselfminimally activated these pathways, was used. Under these conditions,cotransfection of cDNAs encoding full length or the soluble form ofTNF-gamma-beta resulted in significant NF-κB activation. This signalingevent was dependent on the ectopic expression of DR3 and the intactnessof the DR3 death domain, as TNF-gamma-beta alone or in combination witha DD-deleted DR3 did not induce NF-κB activation in these cells.Cotransfection of DR3 with cDNAs encoding TNF-gamma-alpha (full lengthor N-terminal 24-aa truncated) failed to induce NF-κB activation. Asimilar induction of NF-κB activity was observed when increasing amountsof recombinant TL1β protein (aa 72-251, with or without FLAG tag) wereadded to DR3 expressing cells. This induction of NF-κB was specificallyinhibited by the addition of excess amount of DR3-Fc or TR6-Fc, but notby the addition of Fas-Fc or TNFR1-Fc. These results demonstrated thatTNF-gamma-beta is a signaling ligand for DR3 that induces NF-κBactivation, and TR6 can specifically inhibit this event.

[0658] TL1β Induces IL-2 Responsiveness and Cytokine Secretion fromActivated T Cells

[0659] As DR3 expression is mostly restricted to the lymphocytes(Chinnaiyan et al., Science 274:990-2 (1996); Kitson et al., Nature384:372-5 (1996); Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmeret al., Immunity 6:79-88 (1997); Screaton et al., Proc. Natl. Acad. Sci.94:4615-19 (1997); Tan et al., Gene 204:35-46 (1997)) and is upregulatedupon T cell activation, we examined the biological activity ofTNF-gamma-beta on T cells. Recombinant TNF-gamma-beta (aa 72-251)protein was tested for its ability to induce proliferation of resting orcostimulated T cells (treated with amounts of anti-CD3 and anti-CD28that are not sufficient to induce proliferation). In resting orcostimulated T cells, no significant increase in proliferation overbackground was observed. Interestingly, cells that were previouslytreated with TNF-gamma-beta for 72 hours were able to proliferatesignificantly in the presence of IL-2 than cells without TNF-gamma-betapreincubation, indicating that TNF-gamma-beta increases the IL-2responsiveness of costimulated T cells.

[0660] As enhanced IL-2 responsiveness has been associated withincreased IL-2 receptor expression and altered cytokine secretion, itwas of interest to assess these responses on costimulated T cellstreated with TNF-gamma-beta. TNF-gamma-beta treatment indeed upregulatedIL-2Rα (CD25) and IL-2Rβ (CD122) expression from these cells. The extentof the increase in IL-2 receptor expression is consistent with themoderate increase in IL-2 responsiveness compared with IL-2 itself. Wenext measured cytokine secretion from these cells and found that bothIFNγ and GMCSF were significantly induced, whereas IL-2, IL-4, IL-10, orTNF were not. This effect was mostly dependent on the T cell coactivatorCD28, as treatment of the cells with anti-CD3 and TNF-gamma-beta onlyminimally induced cytokine secretion. The effect that we observed on Tcells was specifically mediated by TNF-gamma-beta, as addition ofmonoclonal neutralizing antibody to TL1β, or addition of DR3-Fc orTR6-Fc proteins was able to inhibit TNF-gamma-beta-mediated IFNγsecretion. TNF-gamma-beta was also tested on a variety of primary cells,including B cells, NK cells, and monocytes, but no significant activitywas detected, suggesting a specific activity of TNF-gamma-beta on Tcells.

[0661] TL1β Induces Caspase Activation in TF-1 Cells but not in T Cells

[0662] Overexpression of DR3 in cell lines induces caspase activation(Chinnaiyan et al., Science 274:990-2 (1996); Kitson et al., Nature384:372-5 (1996); Marsters et al., Curr. Biol. 6:1669-76 (1996); Bodmeret al., Immunity 6:79-88 (1997)). We tested whether TL1β could inducecaspase activation in primary T cells. Purified T cells were activatedwith PHA and incubated with recombinant TNF-gamma-beta or FasL in thepresence or absence of cycloheximide (CHX). No induction of caspaseactivity was detected in TNF-gamma-beta treated T cells, but was readilymeasured when cells were triggered with FasL, suggesting that under thisexperimental condition, TNF-gamma-beta does not activate caspases in Tcells (the assay we used detects activation of caspases 2, 3, 6, 7, 8,9, and 10). Various cell lines for the expression of DR3 and found thatthe erythroleukimic cell line TF-1 expressed high levels of DR3 werethen analyzed. The effect of recombinant TNF-gamma-beta protein oncaspase activation in TF-1 cells was then measured. In the absence ofcycloheximide, no significant increase in caspase activity was detectedfollowing TNF-gamma-beta treatment, while TNF-gamma-beta was able toefficiently induce caspase activation in the presence of cycloheximide.This effect was inhibited by either DR3-Fc or TR6-Fc protein but not byLIGHT-Fc. An anti-TNF-gamma-beta monoclonal antibody was also shown tocompletely inhibit this activity, confirming that the caspase activationwas mediated by TNF-gamma-beta.

[0663] TL1β Promotes Splenocyte Alloactivation in Mice

[0664] To determine if the in vitro activities of TNF-gamma-beta couldbe reproduced in vivo, a mouse model of acute graft-versus-host-response(GVHR) was developed in which parental C57BL/6 splenocytes were injectedintravenously into (BALB/c X C57 BU6) F1 mice (CB6F1), and therecipient's immune responses were measured. Typical alloactivationresults in increased splenic weight of the recipient mice and enhancedproliferation and cytokine production of the splenocytes culturedex-vivo (Via, J. Immunol. 146:2603-9 (1991); Zhang et al., J. Clin.Invest. 107:1459-68 (2001)). The large number of T cells in the spleenand their expected upregulation of DR3 in response to alloactivationmakes this an ideal model to assess the effect of TNF-gamma-beta on adefined in vivo immune response. Five day administration of 3 mg/kg ofthe recombinant TNF-gamma-beta protein markedly enhanced thegraft-versus-host responses. The mean (n=4) weight of normal spleensobtained from naive CB6F1 mice was 0.091 g. Alloactivation resulted in a2.5 fold increase in splenic weight (˜0, 228 g). Treatment ofallografted CB6F1 mice with recombinant TNF-gamma-beta protein (aa72-251) further increased splenic weight about 50%, to a mean value of0.349 g. TNF-gamma-beta treatment also significantly enhanced ex-vivosplenocyte expansion, and secretion of IFNγ and GMCSF. Thus,TNF-gamma-beta strongly enhances GVHR in vivo, and this effect isconsistent with TNF-gamma-beta's in vitro activities.

[0665] Experimental Procedures

[0666] Cells, Constructs, and Other Reagents

[0667] All human cancer cell lines and normal lung fibroblast (HFL-1)were purchased from American Tissue Culture Collection. Human primarycells were purchased from Clonetics Corp. Cells were cultured asrecommended. Human cDNA encoding the full length TNF-gamma-alpha,TNF-gamma-beta, DR3; the extracellular domain of TNF-gamma-alpha (aa25-174), TNF-gamma-beta (aa 72-251), BlyS (aa 134-285), FasL (aa130-281), and death domain truncated DR3 (DR3_DD, aa 1-345) wereamplified by PCR and cloned into the mammalian expression vectors pC4and/or pFLAGCMV 1 (Sigma). The extracellular domain of human DR3 (aa1-199), TACI (aa 1-159), HveA (aa 1-192), Fas (aa 1-169), and fulllength TR6 (aa 1-300), was each fused in-frame, at its C-terminus, tothe Fc domain of human IgG1 and cloned into pC4. Rabbit polyclonalTNF-gamma-beta antibody was generated using recombinant TNF-gamma-beta(aa 72-251) protein and purified on a TNF-gamma-beta affinity column.Monoclonal antibodies were raised against recombinant TNF-gamma-beta asdescribed (Kohler and Milstein, Nature 256:503-519 (1975)).

[0668] Cloning of Human, Mouse, and Rat TNF-gamma-beta cDNA

[0669] TNF-gamma-beta was identified by screening a human EST databasefor sequence homology with the extracellular domain of TNF, using theblastn and tblastn algorithms. The extracellular domain of the mouse andrat TNF-gamma-beta cDNA was isolated by PCR amplification from mouse orrat kidney Marathon-Ready cDNAs (Clontech) using human TNF-gamma-betaspecific primers. The resulting sequences were then used to design mouseand rat TNF-gamma-beta specific primers to amplify the 5′ and 3′ ends ofthe cDNA using Marathon cDNA Amplification kit (Clontech). Each sequencewas derived and confirmed from at least two independent PCR products.

[0670] Generation of TNF-gamma-beta Stable Cell line

[0671] HEK293F cells were transiently transfected with pcDNA3.1 (+)(vector control) or pcDNA3. 1 (+) containing full length TNF-gamma-beta.Cells resistant to 0.5 mg/ml Genticin (Invitrogen) were selected andexpanded. Expression of TNF-gamma-beta mRNA was confirmed byquantitative RT-PCR analysis and surface expression of TNF-gamma-betaprotein confirmed by FACS analyses using TNF-gamma-beta monoclonalantibodies.

[0672] Quantitative Real-Time PCR (TaqMan) and RT-PCR Analysis

[0673] Total RNA was isolated from human cell lines and primary cellsusing TriZOL (Invitrogen). TaqMan was carried out in a 25 μl reactioncontaining 25 ng of total RNA, 0.6 μM each of gene-specific forward andreverse primers and 0.2 μM of gene-specific fluorescence probe.TNF-gamma-beta specific primers (forward: 5′-CACCTCTTAGAGCAGACGGAGATAA-3′ (SEQ ID NO:18), reverse: 5′-TTAAAGTGCTGTGTGGGAGTTTGT-3′(SEQ ID NO:19), and probe: 5′-CCAAGGGCACACCTGACAGTTGTGA-3′ (SEQ IDNO:20)) amplify an amplicon span nucleotide 257 to 340 of theTNF-gamma-beta cDNA (aa 86-114 of the protein), while TNF-gamma-alphaspecific primers (forward: 5-CAAAGT CTACAGTTTCCCAATGAGAA-3′ (SEQ IDNO:21); reverse: 5′-GGGAACTGATTTTTA AAGTGCTGTGT- 3′ (SEQ ID NO:22);probe: 5′-TCCTCTTTCTTGTCTTTCCAGTT GTGAGACAAAC-3′ (SEQ ID NO:23)) amplifynucleotide 17 to 113 of the TNF-gamma-alpha cDNA (aa 7-37 of theprotein). Gene-specific PCR products were measured using an ABI PRISM7700 Sequence Detection System following the manufacturer's instruction(PE Corp.). The relative mRNA level of TNF-gamma-beta was normalized tothe 18S ribosomal RNA internal control in the same sample. For RT-PCRanalysis, 0.5 micrograms of total RNA was amplified with TNF-gamma-alpha(5′-GCAAAGTCTACAGTTTCCCAATGAG AAAATTAATCC-3′ (SEQ ID NO:24)) orTNF-gamma-beta specific sense primer (5′-ATGGCCGAGGATCTGGGACTGAGC-3′(SEQ ID NO:25)) and an antisense primer(5′-CTATAGTAAGAAGGCTCCAAAGAAGGTTTTATCTTC-3′ (SEQ ID NO:26)) usingSuperScript One-Step RT-PCR System (Invitrogen). β-actin was used asinternal control.

[0674] Transfection and NF-κB Reporter Assay

[0675] 293T cells were transiently transfected using LipofectAMINE andPLUS reagents according to the manufacturer's instruction (Invitrogen).For reporter assays, 293T cells, at 5×10 cells/well, were seeded in6-well plates and transfected with a total of 1 microgram of DNA. pC4DNA was used as filler DNA. Conditioned supernatant was collected 24hours post-transfection and assayed for secreted alkaline phosphatase(SEAP) activity using the Phospha-Light™ chemiluminescent reporter geneassay system (Tropix). pCMV-lacZ was used as internal control fortransfection efficiency normalization.

[0676] Recombinant Protein Purification

[0677] FLAG fusion proteins were produced from 293T cells by transienttransfection, and purified on anti-Flag M2 affinity columns (Sigma)according to manufacturer's instruction. Receptor proteins with orwithout Fc fusion were produced from Baculovirus or CHO stable celllines as described (Zhang et al., J. Clin. Invest. 107:1459-68 (2001)).Recombinant, untagged TNF-gamma-beta protein (aa 72-251) was generatedand purified from E. coli. Briefly, E. coli cell extract was separatedon a HQ-50 anion exchange column (Applied Biosystems) and eluted with asalt gradient. The 0.2 M NaCl elution was diluted and loaded on a HQ-50column, and the flow through was collected, adjusted to 0.8 M ammoniasulfate and loaded on a Butyl-650s column (Toso Haus). The column waseluted with a 0.6M to 0 M ammonia sulfate gradient and the fractionscontaining TNF-gamma-beta protein were pooled and further purified bysize exclusion on a Superdex-200 column (Pharmacia) in PBS. Allrecombinant proteins were confirmed by NH₂-terminal sequencing on aABI-494 sequencer (Applied Biosystem). The endotoxin level of thepurified protein was less than 10 EU/mg as measured on a LAL-5000E (CapeCod Associates).

[0678] Flow Cytometry, Immunoprecipitation, and Western Blot Analysis

[0679] One million cells, in 0.1 ml of FACS buffer (PBS, 0.1% BSA, 0.1%NaN₃), were incubated with 0.1-1 microgram of protein or antibody at RTfor 15 min. The cells were washed with 3 ml of FACS buffer, reacted withbiotinylated primary antibody, and stained with PE-conjugated secondaryantibody at RT for 15 min. Cells were then washed again, resuspended in0.5 microgram/ml of propidium iodide, and live cells were gated andanalyzed on a FACScan using the CellQuest software (BD Biosciences).

[0680] For coimmunoprecipiation studies, 2 micrograms each of purifiedTNFR-Fc proteins was incubated with 1 microgram of Flag-taggedTNF-gamma-beta, FasL or BlyS protein and 20 microliters of proteinA-Sepharose beads in 0.5 ml of IP buffer (DMEM, 10% FCS, 0.1% TritonX-100) at 4° C. for 4 hr. The beads were then precipitated and washedextensively with PBST buffer (PBS, 0.5% Triton X-100) before boiled inSDS-sample buffer. Proteins were resolved on 4-20% Tris-Glycine gels(NOVEX), transferred to nitrocellulose membranes, and blotted withanti-Flag M2 monoclonal antibody (1 microgram/ml, Sigma) and horseradishperoxidase (HRP)-conjugated goat anti-mouse IgG antibody (0.5microgram/ml).

[0681] BIAcore Analysis

[0682] Recombinant TNF-gamma-beta (from E. coli) binding to varioushuman TNF receptors was analyzed on a BIAcore 3000 instrument. TNFR-Fcwere covalently immobilized to the BIAcore sensor chip (CM5 chip) viaamine groups using N-ethyl-N′-(dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide chemistry. A control receptor surfaceof identical density was prepared, BCMA-Fc, that was negative forTNF-gamma-beta binding and used for background subtraction. Eightdifferent concentrations of TNF-gamma-beta (range: 3-370 nM) were flowedover the receptor-derivatized flow cells at 15 microliters/min for atotal volume of 50 microliters. The amount of bound protein wasdetermined during washing of the flow cell with HBS buffer (10 mM HEPES,pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Surfactant P20). The flow cellsurface was regenerated by displacing bound protein by washing with 20microliters of 10 mM glycine-HCl, pH 2.3. For kinetic analysis, the onand off rates were determined using the kinetic evaluation program inBIAevaluation 3 software using a 1:1 binding model and the globalanalysis method.

[0683] T cell Proliferation Assays

[0684] Whole blood from human donors was separated by Ficoll (ICNBiotechnologies) gradient centrifugation and cells were culturedovernight in RPMI containing 10% FCS (Biofluids). T cells were separatedusing the MACS PanT separation kit (Milteny Biotech), the T cell purityachieved was usually higher that 90%. The cells were seeded on anti-CD3(0.3 microgram/ml, Pharmingen) and anti-CD28 (5.0 microgram/ml) coated96-well plates at 2×10⁴/well, and were incubated with medium alone, 1ng/ml of IL-2 (R & D Systems), or 100 ng/ml of TNF-gamma-beta (aa72-251) at 37° C. After 72 hours in culture, the cells were eitheruntreated or treated with 1 ng/ml of IL-2, and pulsed with 0.5 μCi of³H-thymidine for another 24 hours and incorporation of ³H measured on ascintillation counter.

[0685] Cytokine ELISA Assays for Primary Cells

[0686] 1×10⁵ cells/ml of purified T cells were seeded in a 24-welltissue culture plate that had been coated with anti-CD3 (0.3microgram/ml) and anti-CD28 (5.0 microgram/ml) overnight at 4° C.Recombinant TNF-gamma-beta (aa72-251) protein (100 ng/ml) was added tocells and supernatants were collected 72 hours later. ELISA assay forIFNγ, GM-CSF, IL-2 IL-4, IL-10 and TNFα were performed using kitspurchased from R & D Systems. Recombinant human IL-2 (5 ng/ml) was usedas a positive control. All samples were tested in duplicate and resultswere expressed as an average of duplicate samples plus or minus error.

[0687] Caspase Assay

[0688] TF-1 cells or PHA-activated primary T cells were seeded at 75,000cells/well in a black 96-well plate with clear bottom (Becton Dickinson)in RPMI Medium containing 1% fetal bovine serum (Biowhittaker). Cellswere treated with TNF-gamma-beta (aa72-251, 100 ng/ml) in the presenceor absence of cycloheximide (10 micrograms/ml). Caspase activity wasmeasured directly in the wells by adding equal volume of a lysis buffercontaining 25 μM DEVD-rhodamine 110 (Roche Molecular Biochemicals), andallowed the reaction to proceed at 37C for 1 to 2 hours. Release ofrhodamine 110 was monitored with a Wallac Victor2 fluorescence platereader with excitation filter 485 nm and emission filter 535 nm.

[0689] For the inhibition studies using Fc-proteins or antibodies, theindicated amount of each protein was mixed with either medium or 100ng/ml of TNF-gamma-beta in the presence or absence of cycloheximide. Thereagents were incubated for 1 hour at RT to allow the formation ofprotein- TNF-gamma-beta complexes and then added to the cells. Caspaseactivity was measured as described above.

[0690] Murine Graft-Versus-Host Reaction

[0691] The F1 (CB6F1) of C57BL/6×BALB/c mice (H-2^(bxd)) were transfusedintravenously with 1.5×10⁸ spleen cells from C57BU6 mice (H-2^(b)) onday 0. Recombinant TNF-gamma-beta (aa 72-251) protein or buffer alonewas administered intravenously daily for 5 days at 3 mg/kg/day startingon the same day as the transfusion. The spleens of the recipient F1 micewere harvested on day 5, weighed and single cell suspensions preparedfor in vitro assays.

[0692] Ex-vivo Mouse Splenocyte Alamar Blue and Cytokine Assays

[0693] Splenocytes from normal and the transfused F1 mice were culturedin triplicate in 96-well flat-bottomed plates (4×10⁵ cells/200microliters/well) for 2-4 days. After removing 100 microliters ofsupernatant per well on the day of harvest, 10 microliters Alamar Blue(Biosource) was added to each well and the cells cultured for anadditional 4 hours. The cell number in each well was assessed accordingto OD590 nm minus OD530 nm background, using a CytoFluor apparatus(PerSeptive Biosystems). Cytokines in the culture supernatant weremeasured with commercial ELISA kits from Endogen or R & D Systemsfollowing manufacturer's instructions.

[0694] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0695] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the appended claims.

[0696] The entire disclosures of all patents, patent applications, andpublications referred to herein are hereby incorporated by reference.

1 26 1 1783 DNA Homo sapiens CDS (198)..(1481) 1 catgggtggg ggtgggggcgctgctggatt cctgctctgg tggaggggaa acttgtgagg 60 ggctggtaag cgccccctccgaagcctggt gtgtgcgcgg ggggaaggaa gttagtttcc 120 tctccaccca tgggcaccccttctgcccgg ggcctgggaa gtgggctgct ctgtgggcaa 180 atgctggggc ctctgaa atggag gag acg cag cag gga gag gcc cca cgt 230 Met Glu Glu Thr Gln Gln GlyGlu Ala Pro Arg 1 5 10 ggg cag ctg cgc gga gag tca gca gca cct gtc ccccag gcg ctc ctc 278 Gly Gln Leu Arg Gly Glu Ser Ala Ala Pro Val Pro GlnAla Leu Leu 15 20 25 ctg gtg ctg ctg ggg gcc cgg gcc cag ggc ggc act cgtagc ccc agg 326 Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg SerPro Arg 30 35 40 tgt gac tgt gcc ggt gac ttc cac aag aag att ggt ctg ttttgt tgc 374 Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe CysCys 45 50 55 aga ggc tgc cca gcg ggg cac tac ctg aag gcc cct tgc acg gagccc 422 Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro60 65 70 75 tgc ggc aac tcc acc tgc ctt gtg tgt ccc caa gac acc ttc ttggcc 470 Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala80 85 90 tgg gag aac cac cat aat tct gaa tgt gcc cgc tgc cag gcc tgt gat518 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gln Ala Cys Asp 95100 105 gag cag gcc tcc cag gtg gcg ctg gag aac tgt tca gca gtg gcc gac566 Glu Gln Ala Ser Gln Val Ala Leu Glu Asn Cys Ser Ala Val Ala Asp 110115 120 acc cgc tgt ggc tgt aag cca ggc tgg ttt gtg gag tgc cag gtc agc614 Thr Arg Cys Gly Cys Lys Pro Gly Trp Phe Val Glu Cys Gln Val Ser 125130 135 caa tgt gtc agc agt tca ccc ttc tac tgc caa cca tgc cta gac tgc662 Gln Cys Val Ser Ser Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys 140145 150 155 ggg gcc ctg cac cgc cac aca cgg cta ctc tgt tcc cgc aga gatact 710 Gly Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr160 165 170 gac tgt ggg acc tgc ctg cct ggc ttc tat gaa cat ggc gat ggctgc 758 Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys175 180 185 gtg tcc tgc ccc acg agc acc ctg ggg agc tgt cca gag cgc tgtgcc 806 Val Ser Cys Pro Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala190 195 200 gct gtc tgt ggc tgg agg cag atg ttc tgg gtc cag gtg ctc ctggct 854 Ala Val Cys Gly Trp Arg Gln Met Phe Trp Val Gln Val Leu Leu Ala205 210 215 ggc ctt gtg gtc ccc ctc ctg ctt ggg gcc acc ctg acc tac acatac 902 Gly Leu Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr220 225 230 235 cgc cac tgc tgg cct cac aag ccc ctg gtt act gca gat gaagct ggg 950 Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu AlaGly 240 245 250 atg gag gct ctg acc cca cca ccg gcc acc cat ctg tca cccttg gac 998 Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro LeuAsp 255 260 265 agc gcc cac acc ctt cta gca cct cct gac agc agt gag aagatc tgc 1046 Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys IleCys 270 275 280 acc gtc cag ttg gtg ggt aac agc tgg acc cct ggc tac cccgag acc 1094 Thr Val Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro GluThr 285 290 295 cag gag gcg ctc tgc ccg cag gtg aca tgg tcc tgg gac cagttg ccc 1142 Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp Asp Gln LeuPro 300 305 310 315 agc aga gct ctt ggc ccc gct gct gcg ccc aca ctc tcgcca gag tcc 1190 Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr Leu Ser ProGlu Ser 320 325 330 cca gcc ggc tcg cca gcc atg atg ctg cag ccg ggc ccgcag ctc tac 1238 Pro Ala Gly Ser Pro Ala Met Met Leu Gln Pro Gly Pro GlnLeu Tyr 335 340 345 gac gtg atg gac gcg gtc cca gcg cgg cgc tgg aag gagttc gtg cgc 1286 Asp Val Met Asp Ala Val Pro Ala Arg Arg Trp Lys Glu PheVal Arg 350 355 360 acg ctg ggg ctg cgc gag gca gag atc gaa gcc gtg gaggtg gag atc 1334 Thr Leu Gly Leu Arg Glu Ala Glu Ile Glu Ala Val Glu ValGlu Ile 365 370 375 ggc cgc ttc cga gac cag cag tac gag atg ctc aag cgctgg cgc cag 1382 Gly Arg Phe Arg Asp Gln Gln Tyr Glu Met Leu Lys Arg TrpArg Gln 380 385 390 395 cag cag ccc gcg ggc ctc gga gcc gtt tac gcg gccctg gag cgc atg 1430 Gln Gln Pro Ala Gly Leu Gly Ala Val Tyr Ala Ala LeuGlu Arg Met 400 405 410 ggg ctg gac ggc tgc gtg gaa gac ttg cgc agc cgcctg cag cgc ggc 1478 Gly Leu Asp Gly Cys Val Glu Asp Leu Arg Ser Arg LeuGln Arg Gly 415 420 425 ccg tgacacggcg cccacttgcc acctaggcgc tctggtggcccttgcagaag 1531 Pro ccctaagtac ggttacttat gcgtgtagac attttatgtcacttattaag ccgctggcac 1591 ggccctgcgt agcagcacca gccggcccca cccctgctcgcccctatcgc tccagccaag 1651 gcgaagaagc acgaacgaat gtcgagaggg ggtgaagacatttctcaact tctcggccgg 1711 agtttggctg agatcgcggt attaaatctg tgaaagaaaacaaaacaaaa caaaaaaaaa 1771 aaaaaaaaaa aa 1783 2 428 PRT Homo sapiens 2Met Glu Glu Thr Gln Gln Gly Glu Ala Pro Arg Gly Gln Leu Arg Gly 1 5 1015 Glu Ser Ala Ala Pro Val Pro Gln Ala Leu Leu Leu Val Leu Leu Gly 20 2530 Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg Cys Asp Cys Ala Gly 35 4045 Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys Arg Gly Cys Pro Ala 50 5560 Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro Cys Gly Asn Ser Thr 65 7075 80 Cys Leu Val Cys Pro Gln Asp Thr Phe Leu Ala Trp Glu Asn His His 8590 95 Asn Ser Glu Cys Ala Arg Cys Gln Ala Cys Asp Glu Gln Ala Ser Gln100 105 110 Val Ala Leu Glu Asn Cys Ser Ala Val Ala Asp Thr Arg Cys GlyCys 115 120 125 Lys Pro Gly Trp Phe Val Glu Cys Gln Val Ser Gln Cys ValSer Ser 130 135 140 Ser Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys Gly AlaLeu His Arg 145 150 155 160 His Thr Arg Leu Leu Cys Ser Arg Arg Asp ThrAsp Cys Gly Thr Cys 165 170 175 Leu Pro Gly Phe Tyr Glu His Gly Asp GlyCys Val Ser Cys Pro Thr 180 185 190 Ser Thr Leu Gly Ser Cys Pro Glu ArgCys Ala Ala Val Cys Gly Trp 195 200 205 Arg Gln Met Phe Trp Val Gln ValLeu Leu Ala Gly Leu Val Val Pro 210 215 220 Leu Leu Leu Gly Ala Thr LeuThr Tyr Thr Tyr Arg His Cys Trp Pro 225 230 235 240 His Lys Pro Leu ValThr Ala Asp Glu Ala Gly Met Glu Ala Leu Thr 245 250 255 Pro Pro Pro AlaThr His Leu Ser Pro Leu Asp Ser Ala His Thr Leu 260 265 270 Leu Ala ProPro Asp Ser Ser Glu Lys Ile Cys Thr Val Gln Leu Val 275 280 285 Gly AsnSer Trp Thr Pro Gly Tyr Pro Glu Thr Gln Glu Ala Leu Cys 290 295 300 ProGln Val Thr Trp Ser Trp Asp Gln Leu Pro Ser Arg Ala Leu Gly 305 310 315320 Pro Ala Ala Ala Pro Thr Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro 325330 335 Ala Met Met Leu Gln Pro Gly Pro Gln Leu Tyr Asp Val Met Asp Ala340 345 350 Val Pro Ala Arg Arg Trp Lys Glu Phe Val Arg Thr Leu Gly LeuArg 355 360 365 Glu Ala Glu Ile Glu Ala Val Glu Val Glu Ile Gly Arg PheArg Asp 370 375 380 Gln Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln Gln GlnPro Ala Gly 385 390 395 400 Leu Gly Ala Val Tyr Ala Ala Leu Glu Arg MetGly Leu Asp Gly Cys 405 410 415 Val Glu Asp Leu Arg Ser Arg Leu Gln ArgGly Pro 420 425 3 1254 DNA Homo sapiens CDS (1)..(1251) 3 atg gag cagcgg ccg cgg ggc tgc gcg gcg gtg gcg gcg gcg ctc ctc 48 Met Glu Gln ArgPro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu 1 5 10 15 ctg gtg ctgctg ggg gcc cgg gcc cag ggc ggc act cgt agc ccc agg 96 Leu Val Leu LeuGly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg 20 25 30 tgt gac tgt gccggt gac ttc cac aag aag att ggt ctg ttt tgt tgc 144 Cys Asp Cys Ala GlyAsp Phe His Lys Lys Ile Gly Leu Phe Cys Cys 35 40 45 aga ggc tgc cca gcgggg cac tac ctg aag gcc cct tgc acg gag ccc 192 Arg Gly Cys Pro Ala GlyHis Tyr Leu Lys Ala Pro Cys Thr Glu Pro 50 55 60 tgc ggc aac tcc acc tgcctt gtg tgt ccc caa gac acc ttc ttg gcc 240 Cys Gly Asn Ser Thr Cys LeuVal Cys Pro Gln Asp Thr Phe Leu Ala 65 70 75 80 tgg gag aac cac cat aattct gaa tgt gcc cgc tgc cag gcc tgt gat 288 Trp Glu Asn His His Asn SerGlu Cys Ala Arg Cys Gln Ala Cys Asp 85 90 95 gag cag gcc tcc cag gtg gcgctg gag aac tgt tca gca gtg gcc gac 336 Glu Gln Ala Ser Gln Val Ala LeuGlu Asn Cys Ser Ala Val Ala Asp 100 105 110 acc cgc tgt ggc tgt aag ccaggc tgg ttt gtg gag tgc cag gtc agc 384 Thr Arg Cys Gly Cys Lys Pro GlyTrp Phe Val Glu Cys Gln Val Ser 115 120 125 caa tgt gtc agc agt tca cccttc tac tgc caa cca tgc cta gac tgc 432 Gln Cys Val Ser Ser Ser Pro PheTyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 ggg gcc ctg cac cgc cac acacgg cta ctc tgt tcc cgc aga gat act 480 Gly Ala Leu His Arg His Thr ArgLeu Leu Cys Ser Arg Arg Asp Thr 145 150 155 160 gac tgt ggg acc tgc ctgcct ggc ttc tat gaa cat ggc gat ggc tgc 528 Asp Cys Gly Thr Cys Leu ProGly Phe Tyr Glu His Gly Asp Gly Cys 165 170 175 gtg tcc tgc ccc acg agcacc ctg ggg agc tgt cca gag cgc tgt gcc 576 Val Ser Cys Pro Thr Ser ThrLeu Gly Ser Cys Pro Glu Arg Cys Ala 180 185 190 gct gtc tgt ggc tgg aggcag atg ttc tgg gtc cag gtg ctc ctg gct 624 Ala Val Cys Gly Trp Arg GlnMet Phe Trp Val Gln Val Leu Leu Ala 195 200 205 ggc ctt gtg gtc ccc ctcctg ctt ggg gcc acc ctg acc tac aca tac 672 Gly Leu Val Val Pro Leu LeuLeu Gly Ala Thr Leu Thr Tyr Thr Tyr 210 215 220 cgc cac tgc tgg cct cacaag ccc ctg gtt act gca gat gaa gct ggg 720 Arg His Cys Trp Pro His LysPro Leu Val Thr Ala Asp Glu Ala Gly 225 230 235 240 atg gag gct ctg acccca cca ccg gcc acc cat ctg tca ccc ttg gac 768 Met Glu Ala Leu Thr ProPro Pro Ala Thr His Leu Ser Pro Leu Asp 245 250 255 agc gcc cac acc cttcta gca cct cct gac agc agt gag aag atc tgc 816 Ser Ala His Thr Leu LeuAla Pro Pro Asp Ser Ser Glu Lys Ile Cys 260 265 270 acc gtc cag ttg gtgggt aac agc tgg acc cct ggc tac ccc gag acc 864 Thr Val Gln Leu Val GlyAsn Ser Trp Thr Pro Gly Tyr Pro Glu Thr 275 280 285 cag gag gcg ctc tgcccg cag gtg aca tgg tcc tgg gac cag ttg ccc 912 Gln Glu Ala Leu Cys ProGln Val Thr Trp Ser Trp Asp Gln Leu Pro 290 295 300 agc aga gct ctt ggcccc gct gct gcg ccc aca ctc tcg cca gag tcc 960 Ser Arg Ala Leu Gly ProAla Ala Ala Pro Thr Leu Ser Pro Glu Ser 305 310 315 320 cca gcc ggc tcgcca gcc atg atg ctg cag ccg ggc ccg cag ctc tac 1008 Pro Ala Gly Ser ProAla Met Met Leu Gln Pro Gly Pro Gln Leu Tyr 325 330 335 gac gtg atg gacgcg gtc cca gcg cgg cgc tgg aag gag ttc gtg cgc 1056 Asp Val Met Asp AlaVal Pro Ala Arg Arg Trp Lys Glu Phe Val Arg 340 345 350 acg ctg ggg ctgcgc gag gca gag atc gaa gcc gtg gag gtg gag atc 1104 Thr Leu Gly Leu ArgGlu Ala Glu Ile Glu Ala Val Glu Val Glu Ile 355 360 365 ggc cgc ttc cgagac cag cag tac gag atg ctc aag cgc tgg cgc cag 1152 Gly Arg Phe Arg AspGln Gln Tyr Glu Met Leu Lys Arg Trp Arg Gln 370 375 380 cag cag ccc gcgggc ctc gga gcc gtt tac gcg gcc ctg gag cgc atg 1200 Gln Gln Pro Ala GlyLeu Gly Ala Val Tyr Ala Ala Leu Glu Arg Met 385 390 395 400 ggg ctg gacggc tgc gtg gaa gac ttg cgc agc cgc ctg cag cgc ggc 1248 Gly Leu Asp GlyCys Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly 405 410 415 ccg tga 1254Pro 4 417 PRT Homo sapiens 4 Met Glu Gln Arg Pro Arg Gly Cys Ala Ala ValAla Ala Ala Leu Leu 1 5 10 15 Leu Val Leu Leu Gly Ala Arg Ala Gln GlyGly Thr Arg Ser Pro Arg 20 25 30 Cys Asp Cys Ala Gly Asp Phe His Lys LysIle Gly Leu Phe Cys Cys 35 40 45 Arg Gly Cys Pro Ala Gly His Tyr Leu LysAla Pro Cys Thr Glu Pro 50 55 60 Cys Gly Asn Ser Thr Cys Leu Val Cys ProGln Asp Thr Phe Leu Ala 65 70 75 80 Trp Glu Asn His His Asn Ser Glu CysAla Arg Cys Gln Ala Cys Asp 85 90 95 Glu Gln Ala Ser Gln Val Ala Leu GluAsn Cys Ser Ala Val Ala Asp 100 105 110 Thr Arg Cys Gly Cys Lys Pro GlyTrp Phe Val Glu Cys Gln Val Ser 115 120 125 Gln Cys Val Ser Ser Ser ProPhe Tyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 Gly Ala Leu His Arg HisThr Arg Leu Leu Cys Ser Arg Arg Asp Thr 145 150 155 160 Asp Cys Gly ThrCys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys 165 170 175 Val Ser CysPro Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala 180 185 190 Ala ValCys Gly Trp Arg Gln Met Phe Trp Val Gln Val Leu Leu Ala 195 200 205 GlyLeu Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr 210 215 220Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu Ala Gly 225 230235 240 Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro Leu Asp245 250 255 Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys IleCys 260 265 270 Thr Val Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr ProGlu Thr 275 280 285 Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp AspGln Leu Pro 290 295 300 Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr LeuSer Pro Glu Ser 305 310 315 320 Pro Ala Gly Ser Pro Ala Met Met Leu GlnPro Gly Pro Gln Leu Tyr 325 330 335 Asp Val Met Asp Ala Val Pro Ala ArgArg Trp Lys Glu Phe Val Arg 340 345 350 Thr Leu Gly Leu Arg Glu Ala GluIle Glu Ala Val Glu Val Glu Ile 355 360 365 Gly Arg Phe Arg Asp Gln GlnTyr Glu Met Leu Lys Arg Trp Arg Gln 370 375 380 Gln Gln Pro Ala Gly LeuGly Ala Val Tyr Ala Ala Leu Glu Arg Met 385 390 395 400 Gly Leu Asp GlyCys Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly 405 410 415 Pro 5 455PRT Homo sapiens 5 Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro LeuVal Leu Leu 1 5 10 15 Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val IleGly Leu Val Pro 20 25 30 His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val CysPro Gln Gly Lys 35 40 45 Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys ThrLys Cys His Lys 50 55 60 Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro GlyGln Asp Thr Asp 65 70 75 80 Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr AlaSer Glu Asn His Leu 85 90 95 Arg His Cys Leu Ser Cys Ser Lys Cys Arg LysGlu Met Gly Gln Val 100 105 110 Glu Ile Ser Ser Cys Thr Val Asp Arg AspThr Val Cys Gly Cys Arg 115 120 125 Lys Asn Gln Tyr Arg His Tyr Trp SerGlu Asn Leu Phe Gln Cys Phe 130 135 140 Asn Cys Ser Leu Cys Leu Asn GlyThr Val His Leu Ser Cys Gln Glu 145 150 155 160 Lys Gln Asn Thr Val CysThr Cys His Ala Gly Phe Phe Leu Arg Glu 165 170 175 Asn Glu Cys Val SerCys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180 185 190 Lys Leu Cys LeuPro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser 195 200 205 Gly Thr ThrVal Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu 210 215 220 Leu SerLeu Leu Phe Ile Gly Leu Met Tyr Arg Tyr Gln Arg Trp Lys 225 230 235 240Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys Glu 245 250255 Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser 260265 270 Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr Leu Gly Phe Ser Pro Val275 280 285 Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly AspCys 290 295 300 Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala Pro Pro TyrGln Gly 305 310 315 320 Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser AspPro Ile Pro Asn 325 330 335 Pro Leu Gln Lys Trp Glu Asp Ser Ala His LysPro Gln Ser Leu Asp 340 345 350 Thr Asp Asp Pro Ala Thr Leu Tyr Ala ValVal Glu Asn Val Pro Pro 355 360 365 Leu Arg Trp Lys Glu Phe Val Arg ArgLeu Gly Leu Ser Asp His Glu 370 375 380 Ile Asp Arg Leu Glu Leu Gln AsnGly Arg Cys Leu Arg Glu Ala Gln 385 390 395 400 Tyr Ser Met Leu Ala ThrTrp Arg Arg Arg Thr Pro Arg Arg Glu Ala 405 410 415 Thr Leu Glu Leu LeuGly Arg Val Leu Arg Asp Met Asp Leu Leu Gly 420 425 430 Cys Leu Glu AspIle Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro 435 440 445 Pro Ala ProSer Leu Leu Arg 450 455 6 335 PRT Homo sapiens 6 Met Leu Gly Ile Trp ThrLeu Leu Pro Leu Val Leu Thr Ser Val Ala 1 5 10 15 Arg Leu Ser Ser LysSer Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30 Lys Gly Leu Glu LeuArg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45 Leu Glu Gly Leu HisHis Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60 Pro Gly Glu Arg LysAla Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 Asp Cys Val ProCys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser LysCys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110 Leu Glu ValGlu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125 Cys LysPro Asn Phe Phe Gln Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140 ProCys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150 155160 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 165170 175 Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg180 185 190 Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn GlnGly 195 200 205 Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala IleAsn Leu 210 215 220 Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile AlaGly Val Met 225 230 235 240 Thr Leu Ser Gln Val Lys Gly Phe Val Arg LysAsn Gly Val Asn Glu 245 250 255 Ala Lys Ile Asp Glu Ile Lys Asn Asp AsnVal Gln Asp Thr Ala Glu 260 265 270 Gln Lys Val Gln Leu Leu Arg Asn TrpHis Gln Leu His Gly Lys Lys 275 280 285 Glu Ala Tyr Asp Thr Leu Ile LysAsp Leu Lys Lys Ala Asn Leu Cys 290 295 300 Thr Leu Ala Glu Lys Ile GlnThr Ile Ile Leu Lys Asp Ile Thr Ser 305 310 315 320 Asp Ser Glu Asn SerAsn Phe Arg Asn Glu Ile Gln Ser Leu Val 325 330 335 7 23 DNA ArtificialSequence Description of Artificial Sequence primer 7 gcgccatgggggcccggcgg cag 23 8 30 DNA Artificial Sequence Description of ArtificialSequence primer 8 gcgaagcttc taggacccag aacatctgcc 30 9 33 DNAArtificial Sequence Description of Artificial Sequence primer 9cgcggatccg ccatcatgga ggagacgcag cag 33 10 33 DNA Artificial SequenceDescription of Artificial Sequence primer 10 cgcggatccg ccatcatggagcagcggccg cgg 33 11 54 DNA Artificial Sequence Description ofArtificial Sequence primer 11 gcgtctagat caaagcgtag tctgggacgtcgtatgggta cgggccgcgc tgca 54 12 33 DNA Artificial Sequence Descriptionof Artificial Sequence primer 12 cgcggatccg ccatcatgga ggagacgcag cag 3313 33 DNA Artificial Sequence Description of Artificial Sequence primer13 cgcggatccg ccatcatgga gcagcggccg cgg 33 14 26 DNA Artificial SequenceDescription of Artificial Sequence primer 14 cgcggatcct cacgggccgcgctgca 26 15 33 DNA Artificial Sequence Description of ArtificialSequence primer 15 cgcggatccg ccatcatgga ggagacgcag cag 33 16 33 DNAArtificial Sequence Description of Artificial Sequence primer 16cgcggatccg ccatcatgga gcagcggccg cgg 33 17 35 DNA Artificial SequenceDescription of Artificial Sequence primer 17 gcgagatcta gtctggacccagaacatctg cctcc 35 18 25 DNA Artificial Sequence Description ofArtificial Sequence primer 18 cacctcttag agcagacgga gataa 25 19 24 DNAArtificial Sequence Description of Artificial Sequence primer 19ttaaagtgct gtgtgggagt ttgt 24 20 25 DNA Artificial Sequence Descriptionof Artificial Sequence primer 20 ccaagggcac acctgacagt tgtga 25 21 26DNA Artificial Sequence Description of Artificial Sequence primer 21caaagtctac agtttcccaa tgagaa 26 22 26 DNA Artificial SequenceDescription of Artificial Sequence primer 22 gggaactgat ttttaaagtgctgtgt 26 23 34 DNA Artificial Sequence Description of ArtificialSequence primer 23 tcctctttct tgtctttcca gttgtgagac aaac 34 24 36 DNAArtificial Sequence Description of Artificial Sequence primer 24gcaaagtcta cagtttccca atgagaaaat taatcc 36 25 24 DNA Artificial SequenceDescription of Artificial Sequence primer 25 atggccgagg atctgggact gagc24 26 36 DNA Artificial Sequence Description of Artificial Sequenceprimer 26 ctatagtaag aaggctccaa agaaggtttt atcttc 36

What is claimed is:
 1. A method for treating graft versus host disease,cancer, an immunodeficiency, or an autoimmune disorder comprisingadministering to an individual therapeutically effective amounts of: (a)a first therapeutic agent comprising an antibody which binds to apolypeptide consisting of amino acids 1 to 428 of SEQ ID NO:2 or aminoacids 1 to 417 of SEQ ID NO:4; and (b) a second therapeutic agentselected from the group consisting of: (i) a tumor necrosis factorblocking agent; (ii) an immunosuppressive agent; (iii) an antibiotic;(iv) an anti-inflammatory agent; (v) a chemotherapeutic agent; and (vi)a cytokine.
 2. The method of claim 1, wherein said first therapeuticagent comprises an antibody which binds to a polypeptide consisting ofamino acids 36 to 212 of SEQ ID NO:2.
 3. The method of claim 1, whereinsaid antibody is a monoclonal antibody.
 4. The method of claim 1,wherein said antibody is a polyclonal antibody.
 5. The method of claim1, wherein said antibody is a chimeric antibody.
 6. The method of claim1, wherein said antibody is a humanized antibody.
 7. The method of claim1, wherein said antibody is a single-chain Fv antibody.
 8. The method ofclaim 1, wherein said antibody is an Fab antibody fragment.
 9. Themethod of claim 1, wherein said first and second therapeutic agents areadministered to the individual at the same time.
 10. The method of claim1, wherein said first and second therapeutic agents are administered tothe individual at different times.
 11. The method of claim 1, whereinsaid tumor necrosis factor blocking agent comprises an antibody whichbinds to a protein selected from the group consisting of: (a) TNF-α; (b)TNF-β; and (c) TNF-γ-β.
 12. The method of claim 1, wherein saidimmunosuppressive agent is selected from the group consisting of: (a)cyclosporine; (b) cyclophosphamide; (c) methylprednisone; (d)prednisone; (e) azathioprine; (f) FK-506; and (g) 15-deoxyspergualin.13. The method of claim 1, wherein said cytokine is selected from thegroup consisting of: (a) IL-2; (b) IL-3; (c) IL-4; (d) IL-5; (e) IL-6;(f) IL-7; (g) IL-10; (h) IL-12; (i) IL-13; (j) IL-15; and (k) IFN-γ. 14.A composition comprising: (a) a first therapeutic agent comprising anantibody which binds to a polypeptide consisting of amino acids 1 to 428of SEQ ID NO:2 or amino acids 1 to 417 of SEQ ID NO:4; and (b) a secondtherapeutic agent selected from the group consisting of: (i) a tumornecrosis factor blocking agent; (ii) an immunosuppressive agent; (iii)an antibiotic; (iv) an anti-inflammatory agent; (v) a chemotherapeuticagent; and (vi) a cytokine.
 15. A method for the treatment of graftversus host disease, cancer, an immunodeficiency, or an autoimmunedisorder comprising administering to an individual a therapeuticallyeffective amount of the composition of claim
 14. 16. The composition ofclaim 14 which further comprises a pharmaceutically acceptable carrieror excipient.
 17. An isolated polypeptide comprising an amino acidsequence at least 90% identical to amino acids 36 to 212 of SEQ ID NO:2;wherein said polypeptide is covalently attached to polyethylene glycol,said polyethylene glycol having an average molecule weight selected fromthe group consisting of 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10,000, 15,000, and 20,000.
 18. The isolated polypeptide of claim 17,comprising an amino acid sequence at least 95% identical to amino acids36 to 212 of SEQ ID NO:2.
 19. The isolated polypeptide of claim 18,wherein said amino acid sequence comprises amino acids 36 to 212 of SEQID NO:2.
 20. The isolated polypeptide of claim 17, wherein saidpolypeptide has an average degree of substitution with polyethyleneglycol which falls within a range selected from the group consisting of1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, and 10-12.
 21. Theisolated polypeptide of claim 17, which is produced by a recombinanthost cell.
 22. The isolated polypeptide of claim 17, which comprises aheterologous polypeptide.
 23. A composition comprising the isolatedpolypeptide of claim 17 and a pharmaceutically acceptable carrier. 24.An isolated polynucleotide comprising a nucleic acid encoding an aminoacid sequence at least 90% identical to amino acids 30 to 215 of SEQ IDNO:2.
 25. The isolated polynucleotide of claim 24, comprising a nucleicacid encoding an amino acid sequence at least 95% identical to aminoacids 30 to 215 of SEQ ID NO:2.
 26. The isolated polynucleotide of claim24, comprising a nucleic acid encoding an amino acid sequence at least90% identical to amino acids 30 to 428 of SEQ ID NO:2.
 27. The isolatedpolynucleotide of claim 26, comprising a nucleic acid encoding an aminoacid sequence at least 95% identical to amino acids 30 to 428 of SEQ IDNO:2.
 28. The isolated polynucleotide of claim 27, comprising a nucleicacid encoding amino acids 30 to 428 of SEQ ID NO:2.
 29. The isolatedpolynucleotide of claim 26, comprising a nucleic acid encoding an aminoacid sequence at least 90% identical to amino acids 1 to 428 of SEQ IDNO:2.
 30. The isolated polynucleotide of claim 29, comprising a nucleicacid encoding an amino acid sequence at least 95% identical to aminoacids 1 to 428 of SEQ ID NO:2.
 31. The isolated polynucleotide of claim30, comprising a nucleic acid encoding amino acids 1 to 428 of SEQ IDNO:2.
 32. The isolated polynucleotide of claim 24, further comprising aheterologous polynucleotide.
 33. The isolated polynucleotide of claim32, wherein said heterologous polynucleotide encodes a heterologouspolypeptide.
 34. A method of producing a vector which comprisesinserting the isolated polynucleotide of claim 24 into a vector.
 35. Avector comprising the isolated polynucleotide of claim
 24. 36. Thevector of claim 35, wherein said isolated polynucleotide is operablyassociated with a heterologous regulatory polynucleotide.
 37. A hostcell comprising the isolated polynucleotide of claim
 24. 38. The hostcell of claim 37, wherein said isolated polynucleotide is operablyassociated with a heterologous regulatory polynucleotide.
 39. A methodof producing a polypeptide which comprises culturing the host cell ofclaim 38 under conditions such that said polypeptide is expressed, andrecovering said polypeptide.
 40. An isolated polynucleotide comprising anucleic acid encoding an amino acid sequence at least 90% identical tothe mature amino acid sequence encoded by the cDNA clone in ATCC DepositNo.
 97456. 41. The isolated polynucleotide of claim 40, comprising anucleic acid encoding an amino acid sequence at least 95% identical tothe mature amino acid sequence encoded by the cDNA clone in ATCC DepositNo.
 97456. 42. The isolated polynucleotide of claim 40, comprising anucleic acid encoding an amino acid sequence at least 90% identical tothe complete amino acid sequence encoded by the cDNA clone in ATCCDeposit No.
 97456. 43. The isolated polynucleotide of claim 42,comprising a nucleic acid encoding an amino acid sequence at least 95%identical to the complete amino acid sequence encoded by the cDNA clonein ATCC Deposit No.
 97456. 44. The isolated polynucleotide of claim 40,further comprising a heterologous polynucleotide.
 45. The isolatedpolynucleotide of claim 44, wherein said heterologous polynucleotideencodes a heterologous polypeptide.
 46. A method of producing a vectorwhich comprises inserting the isolated polynucleotide of claim 40 into avector.
 47. A vector comprising the isolated polynucleotide of claim 40.48. The vector of claim 47, wherein said isolated polynucleotide isoperably associated with a heterologous regulatory polynucleotide.
 49. Ahost cell comprising the isolated polynucleotide of claim
 40. 50. Thehost cell of claim 49, wherein said isolated polynucleotide is operablyassociated with a heterologous regulatory polynucleotide.
 51. A methodof producing a polypeptide which comprises culturing the host cell ofclaim 50 under conditions such that said polypeptide is expressed, andrecovering said polypeptide.
 52. An isolated polypeptide comprising anamino acid sequence at least 90% identical to amino acids 30 to 215 ofSEQ ID NO:2.
 53. The isolated polypeptide of claim 52, wherein saidamino acid sequence is at least 95% identical to amino acids 30 to 215of SEQ ID NO:2.
 54. The isolated polypeptide of claim 52, wherein saidamino acid sequence is at least 90% identical to amino acids 30 to 428of SEQ ID NO:2.
 55. The isolated polypeptide of claim 54, wherein saidamino acid sequence is at least 95% identical to amino acids 30 to 428of SEQ ID NO:2.
 56. The isolated polypeptide of claim 55, wherein saidamino acid sequence comprises amino acids 30 to 428 of SEQ ID NO:2. 57.The isolated polypeptide of claim 52, wherein said amino acid sequenceis at least 90% identical to amino acids 1 to 428 of SEQ ID NO:2. 58.The isolated polypeptide of claim 57, wherein said amino acid sequenceis at least 95% identical to amino acids 1 to 428 of SEQ ID NO:2. 59.The isolated polypeptide of claim 58, wherein said amino acid sequencecomprises amino acids 1 to 428 of SEQ ID NO:2.
 60. The isolatedpolypeptide of claim 52, which is produced by a recombinant host cell.61. The isolated polypeptide of claim 52, which comprises a heterologouspolypeptide.
 62. A composition comprising the isolated polypeptide ofclaim 52 and a pharmaceutically acceptable carrier.
 63. An isolatedpolypeptide comprising an amino acid sequence at least 90% identical tothe mature amino acid sequence encoded by the cDNA clone contained inATCC Deposit No.
 97456. 64. The isolated polypeptide of claim 63, whichcomprises an amino acid sequence at least 95% identical to the matureamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo.
 97456. 65. The isolated polypeptide of claim 63, which comprises anamino acid sequence at least 90% identical to the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97456.66. The isolated polypeptide of claim 65, which comprises an amino acidsequence at least 95% identical to the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No.
 97456. 67. Theisolated polypeptide of claim 63, which is produced by a recombinanthost cell.
 68. The isolated polypeptide of claim 63, which comprises aheterologous polypeptide.
 69. A composition comprising the isolatedpolypeptide of claim 63 and a pharmaceutically acceptable carrier. 70.An isolated antibody which binds to a polypeptide consisting of aminoacids 1 to 428 of SEQ ID NO:2.
 71. The isolated antibody of claim 70,wherein said antibody is a monoclonal antibody.
 72. The isolatedantibody of claim 70, wherein said antibody is a polyclonal antibody.73. The isolated antibody of claim 70, wherein said antibody is an Fabantibody fragment.
 74. A method for treating a disease or conditionselected from the group consisting of: (a) cancer; (b) inflammation; (c)an autoimmune disease; and (d) graft v. host disease, wherein saidmethod comprises administering to an individual a therapeuticallyeffective amount of the antibody of claim
 70. 75. A compositioncomprising the antibody of claim 70 and a pharmaceutically acceptablecarrier.
 76. A method for treating an autoimmune disease or preventinggraft rejection in an individual comprising administering to saidindividual a composition of claim
 75. 77. An isolated antibody whichbinds to a polypeptide consisting of the complete amino acid sequenceencoded by the CDNA clone contained in ATCC Deposit No.
 97456. 78. Theisolated antibody of claim 77, wherein said antibody is a monoclonalantibody.
 79. The isolated antibody of claim 77, wherein said antibodyis a polyclonal antibody.
 80. The isolated antibody of claim 77, whereinsaid antibody is an Fab antibody fragment.
 81. A method for treating adisease or condition selected from the group consisting of: (a) cancer;(b) inflammation; (c) an autoimmune disease; and (d) graft v. hostdisease, wherein said method comprises administering to an individual atherapeutically effective amount of the antibody of claim
 77. 82. Acomposition comprising the antibody of claim 77 and a pharmaceuticallyacceptable carrier.
 83. A method for treating an autoimmune disease orpreventing graft rejection in an individual comprising administering tosaid individual a composition of claim 82.